Measuring the velocity of small moving objects such as cells

ABSTRACT

Frequency domain velocity measurements and time domain velocity measurements are made using light from cells or other objects. An optical grating is used to modulate the light from an object so that it has a frequency proportional to the velocity of the object. Depending upon the embodiment, the pitch of the optical grating is uniform or varying. The modulated light is detected, producing an analog signal that is then digitally sampled. Time domain signal processing techniques are used to determine the velocity of the object from the digital samples. Preferably, the velocity measured is applied in determining a timing signal employed for synchronization of an image of the object and an detector signal in an optical analysis system that uses a time delay integration (TDI) detector to determine characteristics of the object in response to light from the object.

RELATED APPLICATION

This application is based on a prior provisional application Ser. No.60/228,076, l filed on Aug. 25, 2000, the benefit of the filing date ofwhich is hereby claimed under 35 U.S.C. §119(e).

FIELD OF THE INVENTION

The present invention generally relates to a method and apparatus formeasuring the velocity of an object, and more specifically, to sensinglight from an object with a light sensitive detector, the amplitude ofthat light signal having been modulated by an optical grating, andmeasuring the velocity of the object by analysis of the modulated lightsignal.

BACKGROUND OF THE INVENTION

Cells and cell groupings are three-dimensional (3D) objects containingrich spatial information. The distribution of a tremendous variety ofbio-molecules can be identified within a cell using an ever-increasingnumber of probes. In the post-genome era there is mounting interest inunderstanding the cell, not only as a static structure, but as a dynamiccombination of numerous interacting feedback control systems. Thisunderstanding can lead to new drugs, better diagnostics, more effectivetherapies, and better health care management strategies. However, thisunderstanding will require the ability to extract a far greater amountof information from cells than is currently possible.

The principal technologies for cellular analysis are automatedmicroscopy and flow cytometry. The information generated by these maturetechnologies, although useful, is often not as detailed as desired.Automated microscopy allows two-dimensional (2D) imaging of from one tothree colors of cells on slides. Typical video frame rates limit kineticstudies to time intervals of 30 ms.

Instruments known as flow cytometers currently provide vital informationfor clinical medicine and biomedical research by performing opticalmeasurements on cells in liquid suspension. Whole blood, fractionatedcomponents of blood, suspensions of cells from biopsy specimens and fromcell cultures, and suspensions of proteins and nucleic acid chains aresome of the candidates suitable for analysis by flow cytometry. In flowcytometers specialized for routine blood sample analysis, cell typeclassification is performed by measuring the angular distribution oflight scattered by the cells and the absorption of light by speciallytreated and stained cells. The approximate numbers of red blood cells,white blood cells of several types, and platelets are reported as thedifferential blood count. Some blood-related disorders can be detectedas shifts in optical characteristics, as compared to baseline opticalcharacteristics, such shifts being indicative of morphological andhistochemical cell abnormalities. Flow cytometers have been adapted foruse with fluorescent antibody probes, which attach themselves tospecific protein targets, and for use with fluorescent nucleic acidprobes, which bind to specific DNA and RNA base sequences byhybridization. Such probes find application in medicine for thedetection and categorization of leukemia, for example, in biomedicalresearch, and drug discovery. By employing such prior art techniques,flow cytometry can measure four to ten colors from living cells.However, such prior art flow cytometry offers little spatial resolution,and no ability to study a cell over time. There is clearly a motivationto address the limitations of existing cell analysis technologies with anovel platform for high speed, high sensitivity cell imaging.

A key issue that arises in cell analysis carried out with imagingsystems is the measurement of the velocity of a cell or other objectthrough the imaging system. In a conventional time-domain methodology,cell velocity is measured using time-of-flight (TOF). Two detectors arespaced a known distance apart and a clock measures the time it takes acell to traverse the two detectors. The accuracy of a TOF measurement isenhanced by increasing detector spacing. However, this increases thelikelihood that multiple cells will occupy the measurement region,requiring multiple timers to simultaneously track all cells in view.Initially, the region between the detectors is cleared before startingsample flow. As cells enter the measurement region, each entry signal istimed separately. The system is synchronized with the sample by notingthe number of entry signals that occur before the first exit signal.

TOF velocity measurement systems are prone to desynchronization when theentry and exit signals are near threshold, noise is present, or expectedwaveform characteristics change due to the presence of different celltypes and orientations. Desynchronization causes errors in velocitymeasurement which can lead to degraded signals and misdiagnosed cellsuntil the desynchronized condition is detected and corrected.Resynchronization may require that all cells be cleared from the regionbetween the detectors before restarting sample flow, causing the loss ofsample.

Significant advancements in the art of flow cytometry are described incommonly assigned U.S. Pat. No. 6,249,341, issued on Jun. 19, 2001, andentitled IMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCHAS CELLS, as well as in commonly assigned U.S. Pat. No. 6,211,955,issued on Apr. 3, 2001, also entitled IMAGING AND ANALYZING PARAMETERSOF SMALL MOVING OBJECTS SUCH AS CELLS. The specifications and drawingsof each of these patents are hereby specifically incorporated herein byreference.

The inventions disclosed in the above noted patents perform highresolution, high-sensitivity 2D and 3D imaging using time delayintegration (TDI) electronic image acquisition with cells in flow. Theseinstruments are designed to expand the analysis of biological specimensin fluid suspensions beyond the limits of conventional flow cytometers.TDI sensors utilize solid-state photon detectors such as charge-coupleddevice (CCD) arrays and shift lines of photon-induced charge insynchronization with the flow of the specimen. The method allows a longexposure time to increase a signal-to-noise ratio (SNR) in the imagewhile avoiding blurring. However, precise synchronization of the TDIdetector timing with the motion of the moving targets is required. Forexample, if a target is to traverse 100 lines of a TDI sensor to buildan image, and the blurring is expected to be less than a single linewidth, then the velocity of the target must be known to less than onepercent of its actual value. It would thus be desirable to providemethod and apparatus capable of producing highly accurate flow velocityfor such moving targets.

Several methods have been suggested in the prior art to address thelimitations of TOF velocity measurements, and to achieve highly accurateflow velocity for moving targets such as cells. One such technique islaser Doppler anemometry (LDA), in which one or more laser beams areused to interrogate a moving target. The Doppler frequency shift isdetected as modulation from the interference of multiple beams that havetraversed different paths in the apparatus. An example of such anapparatus is disclosed in U.S. Pat. No. 3,832,059, issued on Aug. 27,1974, and entitled FLOW VELOCITY MEASURING ARRANGEMENT UTILIZING LASERDOPPLER PROBE. That apparatus employs two laser beams that are directedtoward a moving target at converging angles. The back-scattered lightfrom both beams is collected and focused on a common photodetector.Coherent interference at the detector generates modulation withfrequency equal to twice the Doppler shift frequency, allowing thedetection of the target velocity. While LDA is a functional technique,the LDA systems described in U.S. Pat. No. 3,832,059 are elaborate,expensive, and prone to instability.

In an attempt to overcome the problem of instabilities in the laserwavelength, an improved LDA apparatus is disclosed in U.S. Pat. No.5,229,830, issued on Jul. 20, 1993, and entitled DOPPLER VELOCIMETER. Inthis improved apparatus, a rotating grating is added for the purpose ofextracting diffraction side lobes for use in interrogating the target.This approach eliminates the wavelength dependency in the velocitymeasurement, but adds even more cost and complexity to the velocimeter.

An alternative approach to measuring object velocity by coherent lightinterference is found in LDA instruments that generate fringe patternsin the measurement field. Multiple laser beams create either stationaryor propagating fringe patterns with known periods. Objects crossing thefringes modulate the light collected by the instruments. The modulationfrequency is equal to the velocity divided by the fringe pattern period.LDA instruments of this type are disclosed in U.S. Pat. No. 4,148,585,issued on Apr. 10, 1979, and entitled THREE DIMENSIONAL LASER DOPPLERVELOCIMETER; and in U.S. Pat. No. 5,160,976, issued on Nov. 3, 1992, andentitled OPTICAL DETERMINATION OF VELOCITY USING CROSSED INTERFERENCEFRINGE PATTERNS. The apparatus disclosed in the first of these twopatents uses a rotating diffraction grating and a beam splitter togenerate four interrogation beams, which are then crossed in variouscombinations to measure velocity as a 3D vector. The apparatus disclosedin U.S. Pat. No. 5,160,976 uses specially constructed optical fiberbundles and two lasers of differing wavelength to generate two fringepatterns at the target, such that the patterns are orientedperpendicular to one another, in order to measure velocity as a 2Dvector. Such fringe pattern velocimeters have been adapted for use inmapping complex flow fields. However, the cost, complexity, andinstability inherent in delivering multiple coherent beams to the samplevolume, with tightly constrained wavelengths and alignments, is adeterrent to their use. It would be desirable to provide an alternativemethod and apparatus to measure the velocity of an object entrained in aflow of fluid which is less costly, and more forgiving than these priorsolutions to this problem.

The LDA systems described above rely on the mechanisms of Doppler shiftand wave interference for the measurement of velocity. An alternativeapproach to measuring the velocity of objects in fluids is to collectdata in short bursts at carefully timed intervals, i.e., to collectstroboscopic snapshots of the objects. This approach, which is relatedto but more complicated than the TOF method described above, is calledobject imaging velocimetry (PIV). One such system is disclosed in U.S.Pat. No. 4,729,109, issued on Mar. 1, 1988, and entitled METHOD ANDAPPARATUS FOR MEASURING THE DISPLACEMENTS OF PARTICLE IMAGES FORMULTIPLE EXPOSURE VELOCIMETRY. In this patent, a strobe light sourceilluminates the target field. Cylindrical lenses collapse the image ofthe field into two orthogonal linear projections which are captured bylinear arrays of photodetectors. Signals are collected from the detectorarrays for every flash of the strobe light. For each axis, the signalfrom a first exposure is correlated with the signal from the subsequentexposure to measure target displacement. The displacement is convertedto velocity by dividing the displacement by the time between exposures.The apparatus forms images in which contrast is created by lightabsorption by the target.

A PIV instrument utilizing target fluorescence is disclosed in U.S. Pat.No. 5,333,044, issued on Jul. 26, 1994, and entitled FLUORESCENT IMAGETRACKING VELOCIMETER. In the apparatus described in this patent, aplanar region in the field of flow is illuminated using astrobe-coherent light source. A detector forms 2D images of thefluorescent emission of objects stained with a fluorescent dye. Computeranalysis of the sequence of captured images is used to track the motionof objects in the plane of illumination.

It should be noted that the stabilization and alignment of PIV systemsare less problematic than in LDA systems, but the PIV systems requirepulsed illumination or gated data acquisition to establish timing. ThePIV systems also require arrays of detectors and elaborate data analysisto yield velocity measurements.

A technology that provides a simple, cost-effective alternative to LDAand PIV for measuring object velocity in fluids is based on theinsertion of a grating with alternating opaque and transparent parallelbars in the light path of the photosensor. Light from moving objects ismodulated by the optical grating pattern to create a signal withfrequency directly proportional to the component of velocityperpendicular to the optical grating bars. If object motion isconstrained to this perpendicular direction, then the frequency is equalto the true velocity divided by the period, or pitch, of the opticalgrating. A laser velocimeter based on this principle for measuring thevelocity of a reflective surface moving relative to the instrument isdisclosed in U.S. Pat. No. 3,432,237, issued on Mar. 11, 1969, andentitled VELOCITY MEASURING DEVICE. In the disclosed system of thispatent, the target surface is illuminated with a continuous wave laserand light scattered by the moving surface is collected by a lens andthen delivered to a photosensitive detector through a grating. The barsof the optical grating are oriented perpendicular to the axis of motion.An electronic frequency measuring circuit is used to determine thefrequency of the photosensitive detector. The frequency is conveyeddirectly to a display device for viewing and conversion to velocity.

The application of this method to objects suspended in fluid isdisclosed in U.S. Pat. No. 3,953,126, issued on Apr. 27, 1976, andentitled OPTICAL CONVOLUTION VELOCIMETER. In the disclosed apparatus ofthis patent, light collimated by a lens passes through the flow of fluidand is reflected by a mirror with alternating bars of reflective andabsorptive material. The reflective bars return light through the flowof fluid to be collected by the lens. The lens focuses the reflectedlight on a photosensitive detector. An electronic circuit is used toestimate the frequency of the detector signal and to deliver thefrequency to a display device for viewing.

It should be noted that the hardware signal processors used in earlyimplementations of laser velocimeters have largely been displaced bycomputation-based digital signal processors. The demands on thephotosensor signal processors vary with the nature of the application,but the most stringent applications demand high speed and high accuracy,under conditions of low SNR and rapidly varying flow velocity.

An example of an effective method for extracting velocity from thephotosensor signal of a grating-based laser velocimeter is disclosed inU.S. Pat. No. 5,859,694, issued on Jan. 12, 1999, and entitled OPTICALVELOCIMETER PROBE. In this patent, the digitized photosensor signal iscaptured in blocks of samples for processing. For each block, the signalprocessor executes the steps of generating a complex signal using theHilbert transform, autocorrelating the complex signal, and extractingthe phase for each time sample of the autocorrelogram. Theautocorrelation is performed using the steps of a complex Fouriertransformation, squaring the magnitude of the spectrum, and thenapplying an inverse Fourier transformation. Finally, an optimizationroutine finds a best-fit velocity value for the phase samples. Themethod described in this patent has the advantage of building SNR anddelivering accurate velocity estimates, given long signal segments.However, the method is computation intensive, limiting the rate at whichthe velocity estimate is updated.

It would be desirable to utilize the principal of modulation of lightfrom moving objects by the insertion of a periodic grating into thedetector path for the purpose of measuring object velocity, and tofurther employ improved signal processing, superior control systemdesign, and/or a unique grating design to achieve high precisionvelocity measurements in an imaging flow cytometer.

SUMMARY OF THE INVENTION

The present invention is directed to a system and method for determininga velocity of an object. This method will preferably be employed inconjunction with an imaging or optical system that is adapted todetermine one or more characteristics of an object from an image of theobject or light from the object. There is relative movement between theobject and the imaging system, and although it is contemplated thateither, or both, the object and imaging system may be in motion, theobject will preferably be in motion and the imaging system will befixed. When used in conjunction with an imaging system that incorporatesa TDI detector, the velocity determined according to the presentinvention is used to provide a clocking signal to synchronize the TDIdetector with the movement of the image over the TDI detector.

It should be understood that while portions of the followingdescription, and the claims that follow, refer to “an object,” it isclearly contemplated that the present invention is intended to be usedwith a plurality of objects, and is particularly useful in connectionwith imaging a stream of objects. In at least one embodiment, the streamof objects comprises moving cells or cell clusters. Furthermore, whilethe examples cited herein primarily relate to measuring the velocity ofobjects such as cells flowing in a stream of liquid, the presentinvention applies as well to objects attached to a solid substratemoving relative to the imaging system and to particles or liquiddroplets flowing in a stream of gas.

Fundamental to the present invention are the illumination of an object,and the detection of light from the object. For the purpose of velocitymeasurement, the detected light must carry information about the changeof position of the object over time. The object may modify theillumination light by scattering, refraction, diffraction, or absorptionso that its wavelength is the same as that of the light received by thedetector. Alternatively, fluorescence or phosphorescence at the objectcaused by the illumination light may result in light of differentwavelengths than the illumination light to be received at the detector.Furthermore, the object may emit light used to determine the velocity ofthe object, without requiring prior illumination of the object byanother light source.

The basic velocity measuring system includes “a sensitive volume,”through which objects, preferably entrained in a fluid, are illuminated.Light from the objects is directed along a collection path. Thecollected light is first converted into an electrical signal, and thenthe electrical signal is digitized. The digitized signal is analyzed toextract the velocity of an object. Thus, the velocity measuring systemwill preferably include a light source that illuminates the objects inmotion (unless the object self-emit light without any need forillumination by a light source), a light sensitive detector forreceiving light from the objects, and electronic components tomanipulate the signal from the light sensitive detector to determine avelocity. The light source may be a laser, a light emitting diode, afilament lamp, or a gas discharge arc lamp, for example, combined withoptical conditioning elements such as lenses, apertures, and filters todeliver the desired wavelength(s) of light to the object at an intensityrequired for determination of the velocity of the object. The detectormay be a photomultiplier tube or a solid-state photodetector and may becombined with optical conditioning elements such as lenses, apertures,and filters to deliver the desired wavelengths of light from the objectto the detector.

Light from the object is received as the object passes through thesensitive volume. Because the sensitive volume is bounded by a profileof an illumination field and by an acceptance window of the lightsensitive detector, it would be possible to estimate the velocity of theobject from the time required for the object to pass through thesensitive volume. However, the sensitive volume is bounded by gradientsrather than distinct edges, and it may be impractical to maintain thedimensions of the sensitive volume with a high degree of accuracy.

To address the above-noted problem, one or more optical gratings is usedto establish a distance measurement scale for computing velocity,eliminating the concern for maintaining the dimensions of the sensitivevolume with a high degree of accuracy. The optical gratings arepreferably fabricated using high precision methods, such asphotolithographic etching, to create patterns of alternating bars ofopaque and transparent material. The accuracy required for thefabrication of the optical gratings is a function of the accuracyrequired in the velocity measurement. Preferably the optical gratingincludes alternating sequences of opaque strips and transparent stripsof substantially equal width.

As noted above, when the velocity measurement system is used inconjunction with an imaging system that preferably employs a TDIdetector. In TDI imaging, the charge in a TDI detector is moved insynchrony with an image that is incident on the TDI detector, enabling athousand-fold increase in integration times over conventional frameimaging. TDI imaging is conventionally performed by clocking a pixeloutput from the detector using an encoder that directly measures samplemovement, keeping the charge on the detector synchronized with theimage. Since the transport of objects by a fluid stream prevents the useof an encoder, systems imaging objects in flow require an alternativemeans of synchronizing the TDI detector with the moving object and thus,with the moving image of the object across the TDI detector. The presentinvention provides an acceptable approach for measuring the velocity ofmoving objects (such as cells) for use in such TDI based imagingsystems.

The present invention encompasses two different configurations ofoptical gratings, and two different signal processing methods, which areused to analyze the modulated signal produced by a detector in responseto the modulated light produced by an optical grating, thereby enablingthe velocity of an object to be determined. Preferably, each signalprocessing embodiment is executed under the control of a supervisoryprogram appropriate to the particular method employed.

In one embodiment, the signal is homodyned to a baseband and thepassband is limited to improve the SNR. The frequency of the modulatedlight is extracted in the time domain from signal packets selected forlow phase interference. This method is well suited for execution in afast pipeline processor and delivers relatively high accuracy andrelatively high sensitivity. Another embodiment employs two opticalgratings displaced from one another along the flow axis to modulatelight from the object, so that the modulated light is incident on twodifferent detectors. Cross-correlation of the two signals from thedetectors rapidly delivers a highly accurate estimate of velocity of theobject. The performance of these two embodiments is preferably optimizedby the inclusion of supervisor programs, which rapidly adapt to changingflow velocity and varying signal strength from the detector(s). Thechoice of the embodiment that is employed will depend on the particulardemands of an application for determining the velocity of an object.

More specifically, a velocity measurement system in accord with thepresent invention includes an optical element disposed so that lighttraveling from an object passing through a sensitive volume is directedby an optical element along a collection path. At least one opticalgrating is disposed in the collection path and modulates the light,producing modulated light having a modulation frequency corresponding toa velocity of the object. At least one light sensitive detector isdisposed in the collection path and converts the modulated light into anelectrical signal. The velocity measurement system also includes meansfor converting the electrical signal into a sequence of digital samples,and means for processing the sequence of digital samples to determine avelocity of the object corresponding to the electrical signal.

Preferably, the means for converting the electrical signal into thesequence of digital samples comprises at least one analog-to-digitalconverter. It is likely that most such systems will include at least oneamplifier electrically coupled each light sensitive detector, foramplifying the electrical signal before conversion of the electricalsignal into a sequence of digital samples. In several embodiments,bandpass filters are employed to filter the electrical signal beforeconversion into a sequence of digital samples. Alternatively, theelectrical signal may be converted into a sequence of digital samplesimmediately after amplification, and a digital bypass filter may beutilized. The means for processing the sequence of digital samples cancomprise a computer, an application specific integrated circuit (ASIC),or a digital oscilloscope.

Preferably, a velocity measurement system in accord with the presentinvention will further include a system controller for controlling theacquisition of the electrical signal, for controlling the means forconverting the electrical signal into a sequence of digital samples, andfor controlling the means for processing the sequence of digitalsamples. The system controller is preferably a computer or otherprogrammed computing device, however, it should be understood that anASIC can also be beneficially employed as a system controller.Preferably the system controller will regulate a gain of an amplifierthat amplifies the electrical signal, regulate a threshold applied tofrequency measurements so that measurements made under the condition ofinadequate SNR are rejected, and/or regulate a frequency of a pair ofbaseband converter local oscillators in response to variations in avelocity of the object.

The light from an object passing through the sensitive volume caninclude light scattered by that object, an unstimulated emission fromthe object, or a stimulated emission from the object. While it iscontemplated that the velocity measuring system and method of thepresent invention can use ambient light, at least one light source ispreferably incorporated into such a system for illuminating thesensitive volume (unless the object self-emits light without requiringprior illumination from another source). Such a light source can alsostimulate a fluorescent emission from an object passing through thesensitive volume. Incident light can be at least partially absorbed byan object passing through the sensitive volume, so that the lightdirected by the optical element will have been affected by theabsorption of light by the object. Alternatively, incident light isreflected from an object passing through the sensitive volume toward theoptical element. The light source can be one or more of a coherent lightsource, a non-coherent light source, a pulsed light source, a continuouslight source, a continuous wave laser, a pulsed laser, a continuous waveincandescent lamp, a strobe arc lamp, and may include an optical filterfor selecting a limited spectrum for illumination.

Velocity measurement systems in accord with the present inventionpreferably enable a flow of fluid, in which objects are entrained, topass through the sensitive volume, such that a velocity of an object soentrained can be measured. In other embodiments, velocity measurementsystems in accord with the present invention enable a support, on whichobjects are disposed, to pass through the sensitive volume, such that avelocity of the objects are disposed, to pass through the sensitivevolume can be measured. At least one embodiment will comprise astage-based motion system with a high-resolution linear encoder.

It is anticipated that velocity measuring systems, or combined velocitymeasurement and imaging systems can beneficially include a mechanism forsorting objects disposed downstream of the sensitive volume(s).

In one embodiment in which the optical grating has a uniform pitch, themeans for converting the electrical signal into a sequence of digitalsamples includes an amplifier that produces an amplified electricalsignal based on the electrical signal received from the detector. Themeans also includes a bandpass filter for filtering the amplifiedelectrical signal to produce a passband signal. The means furtherincludes a baseband converter for converting the passband signal into apair of signals that represent the passband signal with two components,a real component and an imaginary component. Each of the two basebandsignals is converted to a sequence of digital samples by ananalog-to-digital converter.

For embodiments wherein the at least one optical grating has a uniformpitch, the means for processing the sequence of digital samplespreferably determines the velocity from a first derivative of a timeseries of phase samples of the electrical signal, after calculatingthose phase samples from a baseband complex representation of theelectrical signal. This processing of the electrical signal can beexecuted by a programmed computer, an ASIC, other hardwire logiccircuits, or a digital oscilloscope.

In another embodiment, two substantially identical optical gratings,each having a non-uniform pattern of opaque bar and transparent gapwidths, are disposed along an axis of motion of an object such that theobject sequentially traverses the sections.

In one preferred embodiment that incorporates non uniform opticalgratings, a beam splitter is disposed in the collection path, such thata portion of the light traveling from an object along the collectionpath is diverted along a second collection path. A second non uniformoptical grating is disposed in the second collection path, such that thesecond optical grating also modulates the light, producing modulatedlight have a modulation frequency corresponding to a velocity of anobject passing through the sensitive volume. A second light sensitivedetector is disposed in the second collection path, and converts thelight modulated by the second optical grating into an electrical signal.

In an embodiment incorporating two optical gratings with a non uniformpitch, the means for converting the electrical signal into a sequence ofdigital samples includes a first amplifier coupled to the detector thatis disposed in the collection path. The first amplifier amplifies theelectrical signal from a first light sensitive detector, producing anamplified electrical, and a first bandpass filter filters the amplifiedelectrical signal to produce a first passband signal. A firstanalog-to-digital converter converts the passband signal to a firstsequence of digital samples. The means for converting the electricalsignal into a sequence of digital samples also includes a secondamplifier disposed in a second collection path. The second amplifieramplifies the second electrical signal, producing a second amplifiedelectrical signal that is filtered by a second bandpass filter,producing a second passband signal. The second passband signal is inputto a second analog-to-digital converter, which converts the secondpassband signal to a second sequence of digital samples. Alternatively,the bandpass filter operation can be accomplished using digital bandpassfilters following the analog-to-digital converters.

Preferably when two non uniform optical gratings are employed, eachoptical grating is aligned in series along an axis of motioncorresponding to an object that the light is collected from, and themeans for processing the sequence of digital samples to determine avelocity of an object calculates an amplitude peak of across-correlogram generated by a convolution of the electrical signalfrom the first light sensitive detector disposed in the collection pathand the second electrical signal from the second light sensitivedetector disposed in the second collection path.

Embodiments with optical gratings of non uniform pitch also preferablyinclude a control system controllably connected to the means forconverting the electrical signal into a sequence of digital samples andthe means for processing the sequence of digital samples. The controlsystem preferably regulates the gain of each amplifier in response tovarying electrical signal levels, and regulates an upper and a lowerlimit for time shifting the cross-correlation step in response tovariations in the velocity of an object.

As noted above, velocity measurement systems in accord with the presentinvention can be incorporated into imaging systems that also determinenon-velocity characteristics of an object passing through the sensitivevolume, by employing at least one TDI detector that using the velocityfor a clocking function. Such combination velocity measurement andimaging systems preferably include a second optical element disposed sothat light traveling from an object passing through a second sensitivevolume passes through the second optical element and travels along yetanother collection path, and at least one additional detector thatdetermines a non-velocity related characteristic of an object passingthrough the second sensitive volume. Preferably, the second sensitivevolume, the second optical element, and the at least one additionaldetector are disposed downstream from the sensitive volume, opticalelement, and light sensitive detector of the velocity measuring portionof the combined system.

It is anticipated that when the objects in question are cells, theaccurate determination of cell velocity for use in a TDI-based imagingsystem for cell analysis, in accord with the present invention, willgreatly expand the amount of information that can be extracted fromcells. It is expected that the present invention will enable systems toprocess sample sizes of about 100 million cells, and analyze the sampleswith high spectral and spatial resolution, at very high speeds. Such asystem will have clinical applications that include non-invasiveprenatal diagnosis via maternal peripheral blood, routine cancerscreening and eventually, therapeutic applications involving theisolation, modification and re-implantation of rare cells. In addition,the present invention is directed to a general purpose velocitydetection method that can be applied to other applications where anaccurate determination of the velocity of an object or the velocity of aflow is required.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same becomesbetter understood by reference to the following detailed description,when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a schematic diagram of a system for measuring the velocity ofobjects in a flow stream by detecting light scattered by the objects;

FIG. 2 is a schematic diagram of a system for measuring the velocity ofobjects in a flow stream by detecting light emitted by fluorescence bythe objects;

FIG. 3 is a schematic diagram of a system for measuring the velocity ofobjects in a flow stream by detecting the absorption of light by theobjects;

FIG. 4 is a schematic illustration of the concept of building a signalfrom the passage of the image of a bright object across a grating;

FIG. 5 is a schematic diagram showing the integration of an opticalgrating into a flow velocity measurement system;

FIG. 6 is a block diagram illustrating the stages of processing thesignal from a light sensitive detector for the purpose of objectvelocity measurement;

FIG. 7A illustrates a graph of a typical photodetector signal beforebandpass filtering;

FIG. 7B illustrates a graph of a typical photodetector after bandpassfiltering;

FIG. 8 is a schematic representation illustrating the operation of a TDIdetector;

FIG. 9 is a schematic diagram of a flow imaging system including a flowvelocity measurement system delivering timing to the TDI detector;

FIG. 10 is a block diagram of the structure of a TDI detector and theassociated subsystems of the flow imaging system;

FIG. 11 is a schematic diagram of a flow imaging system in which objectvelocity measurement is performed upstream from image acquisition;

FIG. 12 is a schematic diagram of a cell sorting apparatus including theelements of a velocity measurement system controlling a droplet chargingsystem;

FIG. 13 is a block diagram illustrating a first embodiment of afrequency domain velocity measurement system in which objects are movedthrough a FOV on a support;

FIG. 14A is an image of beads, ruling, and an adjustable slit acquiredby inserting a beam splitter, lens, and detector after the ruling,placing a light source behind the slide, and opening the slit forclarity;

FIG. 14B is a scattered light image with the slit of FIG. 2A closed downto 200 microns (40 microns in object space) and with the beads movingacross the field of view at 20 mm/s, while data was acquired forapproximately one second;

FIG. 15 is a graph illustrating experimental results for a closed-loopDC servo driven stage that drives 7.9 microns beads affixed to amicroscope slide at 20 mm/s;

FIG. 16 is a graph of showing experimental results comparing commandedvelocity to measured velocity, for the FDVM and encoder;

FIG. 17 is a graph of experimental results showing the linearity of thecommanded velocity and measured velocity, for the FDVM and encoder;

FIG. 18 is a TDI image captured using frequency domain velocityfeedback;

FIG. 19 is a block diagram of common components of flow velocitymeasurement systems, in accord with the present invention that are underthe control of a system controller;

FIG. 20 is a block diagram illustrating the steps of the signalprocessing and velocity computation for a second preferred embodiment ofthe present invention;

FIG. 21 is a block diagram illustrating the steps comprising the signalprocessing for the embodiment of FIG. 20;

FIG. 22 is a graph of an exemplary spectrum of an unmodulatedphotosensor signal for a single object;

FIG. 23 is an enlarged view of the graph of FIG. 22, illustrating thesignal peak;

FIG. 24 is a block diagram illustrating the steps employed by thesupervisory program for controlling the second embodiment of the presentinvention;

FIG. 25 is a block diagram of a double sideband receiver for use in athird embodiment of the present invention;

FIG. 26 schematically illustrates a modification of the spectrum of anexemplary photosensor signal by a baseband converter;

FIG. 27 schematically illustrates a modification of the spectrum of theexemplary photosensor signal of FIG. 26 by a baseband converter;

FIG. 28 schematically illustrates an analysis of the magnitude and phaseseries of the exemplary photosensor signal of FIG. 26 by computations onthe I and Q baseband signals;

FIG. 29 schematically illustrates an application of a phase unwrappingalgorithm to the phase series of the exemplary photosensor signal ofFIG. 26, to provide a monotonic phase series;

FIG. 30 is a block diagram illustrating the steps employed in the phaseunwrapping algorithm;

FIG. 31A is a graph showing a magnitude threshold being applied to asignal representing the monotonic phase series of FIG. 29, to reduce theeffects of random noise;

FIG. 31B is a graph showing the result of employing the magnitudethreshold of FIG. 31A before computing the fractional frequency from themonotonic phase series of FIG. 29;

FIG. 32 is a block diagram of the signal processing and velocitycomputation steps for the third embodiment of the present invention;

FIG. 33 schematically illustrates a computational modification of thespectrum of I and Q baseband signals to provide upper and lower sidebandsignals;

FIG. 34 is a block diagram illustrating the steps comprisingsegmentation and analysis of objects for the third embodiment of thepresent invention;

FIG. 35A is a graph illustrating the summation of the baseband frequencyand the local oscillator frequency;

FIG. 35B is a graph illustrating the conversion of the sum of FIG. 35Ato a velocity;

FIG. 36 is a block diagram illustrating the steps employed by asupervisory program for controlling the third embodiment of the presentinvention;

FIG. 37 is a graph of the sum of the upper sideband power and the lowersideband power for a broad sweep of the local oscillator frequency;

FIG. 38 is a graph showing the transition of power from the uppersideband to the lower sideband for a narrow sweep of the localoscillator;

FIG. 39 schematically illustrates the convolution of two signalsgenerated by a conventional optical grating of uniform pitch;

FIG. 40 schematically illustrates the design of a conventional opticalgrating and its alignment to the Gaussian profile of the illuminationbeam;

FIG. 41 schematically illustrates the design of an optical grating withlinearly swept pitch and its alignment to the Gaussian profile of theillumination beam;

FIG. 42 schematically illustrates the convolution of two signalsgenerated by an optical grating having a linearly swept pitch;

FIG. 43 is a schematic diagram of a velocity measurement system usingstacked gratings of non-uniform pitch, in accord with a fourthembodiment of the present invention;

FIG. 44 schematically illustrates the alignment of images of twogratings of non-uniform pitch relative to the Gaussian beam profile ofthe illumination beam;

FIG. 45 schematically illustrates the convolution of signals from twophotosensors using the stacked non-uniform gratings of the fourthembodiment of the present invention;

FIG. 46 is a graph of an expanded correlogram for the signals generatedby the stacked non-uniform gratings;

FIG. 47 is a block diagram broadly illustrating the steps required forsignal processing and velocity computation in accord with the fourthembodiment of the present invention;

FIG. 48 is a block diagram illustrating detailed steps for theprocessing of a signal segment of the fourth embodiment of the presentinvention;

FIG. 49 schematically illustrates the concept of the convolution of afirst signal by a second similar but delayed signal; and

FIG. 50 is a block diagram illustrating the logical steps implemented bya supervisory program for controlling the fourth embodiment of thepresent invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT Overview of the PresentInvention

In the present invention, moving objects are illuminated and light fromthe objects is imaged onto a detector after passing though an opticalgrating. The optical grating comprises a plurality of transparent andopaque bars that modulate the light received from the object, producingmodulated light having a frequency of modulation that corresponds to thevelocity of the object from which the light was received. Preferably theoptical magnification and the ruling pitch of the optical grating arechosen such that the bars are approximately the size of the objectsbeing illuminated. Thus, the light collected from cells or other objectsis alternately blocked and transmitted through the ruling of the opticalgrating as the object traverses the interrogation region, i.e., the FOV.The modulated light is directed toward a light sensitive detector,producing a signal that can be analyzed by a processor to determine thevelocity of the object.

The present invention has been developed as four distinct preferredembodiments. First and second embodiments employ a first optical gratingand a frequency domain velocity measurement based signal processingtechnique. A third embodiment also employs the first optical grating,but uses a time domain velocity measurement (TDVM) based signalprocessing technique. A fourth embodiment employs the first and alsoincludes a second optical grating to determine velocity using the timedomain based signal processing technique. The differences in the firstand second embodiments are that the first embodiment is specificallydirected to analyzing objects that are deposited on a support that ismoved through a FOV, while the second embodiment applies the samegeneral processing technique to determine the velocity of objects thatare entrained in a fluid flow through the FOV. Details of these specificembodiments are provided below, after a brief discussion of the conceptsgenerally applicable to all of the embodiments.

The present invention can be used with any of the various illuminationand light collection configurations illustrated in FIGS. 1, 2, and 3.However, those configurations should not be considered limiting on thescope of the invention, and are provided merely as exemplaryconfigurations. Each Figure shows a light source, objects in motion(preferably objects entrained in a flow of fluid) illuminated by thelight source, and a velocity detector for receiving light from theobjects. The light source may be a laser, a light emitting diode, afilament lamp, or a gas discharge arc lamp, and the system may includeoptical conditioning elements such as lenses, apertures, and filtersthat are employed to deliver one or more desired wavelengths of light tothe object with an intensity required for detection of the velocity (andoptionally, one or more other characteristics of the object). Thevelocity detector includes a light sensitive detector (not separatelyshown in these figures) comprising, for example, a photomultiplier tubeor a solid-state photodetector, and one or more other opticalconditioning elements such as a lens, aperture, and/or filter, todeliver the modulated light to the light sensitive detector (also notseparately shown in these figures).

FIG. 1 illustrates the configuration of a system 10 a that employs lightscattered by objects 18 traveling through a flow tube 16. An angle 20(designated as angle θ) between the beam axis of an illuminator 12 andan acceptance axis of a velocity detector 14 may be adjusted so thatlight scattered from the objects is delivered to the velocity detector,for a particular scatter angle. The intensity profile of the scatteredlight is a function of the ratio of the size of the scattering elementsto the wavelength of the incident light. A relatively large number ofscattering elements may be present in/on the object, and angle θ may beadjusted for the detection of scattered light from elements within adesired size range for the elements.

FIG. 2 illustrates the configuration of a system 10 b that uses lightemitted by objects 18 traveling in flow tube 16, in response to theabsorption by the objects of light from illuminator 12. In this case,the detection axis will typically be orthogonal to the illumination axisin order to minimize the amount of incident light reaching velocitydetector 14. Typically, a filter or a set of filters (not separatelyshown) will be included in the velocity detector to deliver to the lightsensitive detector only a narrow band of wavelengths of the lighttraveling along a detection path 22 corresponding, for example, to thewavelengths emitted by the fluorescent or phosphorescent molecules inthe object, so that light in the wavelength(s) provided by theilluminator 12 is substantially eliminated.

FIG. 3 illustrates the configuration of a system 10 c utilizing lightfrom illuminator 12 that continues to propagate towards velocitydetector 14 along the axis of illumination; this light is modified byobjects 18 traveling in flow tube 16 by absorption, diffraction, orrefraction. Note that system 10 c is not well adapted for the detectionof light emitted by fluorescence or phosphorescence, due to the highintensity of light emitted by illuminator 12 relative to the intensityof light emitted from objects 18, and that both types of light followthe same path to velocity detector 14. Modification of the light by theobjects caused by absorption can be detected by measuring the intensityof the light incident on the velocity detector. A system of lenses maybe used to restrict the origin of the collected light to a desired fieldin the path of the stream of objects. Modification of the light by theobjects caused by diffraction or refraction may be detected through theuse of a phase contrast method, in which only light subjected to phasemodification by an object is visible, any unmodified light having beencanceled by interference with a reference beam (not separately shown).

In each of the above-noted configurations, the light received by thevelocity detector is modified by objects passing through a FOV. Becausethis FOV is bounded by the profile of the illumination field and by theacceptance window of the velocity detector, it would seem to be possibleto estimate object velocity from the time it takes for the object topass through the FOV. However, the FOV is bounded by gradients ratherthan distinct edges, and it will likely be impractical to maintain thedimensions of the FOV with a high degree of accuracy. This limitation isparticularly true when the objects being illuminated or emitting thelight that is detected are small in size, such as biological cells.

Placing an optical grating in the path of light incident on the velocitydetector establishes a highly precise distance scale for measuringobject velocity. The optical grating concept is illustrated in FIG. 4.This figure shows the positive contrast of the object relative to abackground, which occurs if the light being detected is emitted from theobject by fluorescence or phosphorescence, or is light that is scatteredby the object. Preferably, in the optical grating shown, the opaque andtransparent bar widths are substantially equal and substantially equalto an average diameter of the objects whose velocity is to be measured.This matching of the optical grating to the object size increases thelight modulation amplitude, but it should be noted that the opticalgrating will provide the desired modulating function over a wide rangeof object size relative to the optical grating pitch.

FIG. 4 includes shows four snapshots 24 a- 24 d of an optical grating26, at equally spaced sample times, t₁-t₄, and a signal amplitude 28incident on the light sensitive detector at those times. Note that eachgrating 26 includes alternating opaque zones 34 and transparent zones 32of uniform pitch. The passage of the light emitting object 30 across oneof the transparent zones 32 in the optical grating causes the amplitudeto increase as the exposed area of the object increases, and then todecrease as the object moves behind one of opaque zones 34. In the idealcase, only direct light from objects would reach the detector.Typically, however, some scattered light or light from strayfluorescence will continuously pass through the transparent zones,creating a constant bias or offset in the light sensitive detectoroutput signal.

FIG. 5 shows an optical system 10 d that illustrates how the opticalgrating-based detection system might be implemented for the case inwhich the objects emit photons through the process of fluorescence. Alens 36 creates a focused illumination field at a wavelength λ₁ in a FOV38. Fluorescence in an object 18 a caused by this illumination resultsin photons being emitted by the object collected by lens 40 on thedetector axis. An emission filter 42 is used to reject light atwavelengths other than λ₂. Lens 40 and a lens 44 create a focused imageof the object on an optical grating 46. It would be possible to generatea conjugate image of the object and the optical grating at a camera, inwhich case the camera would produce well-focused images of objectspassing across the sharp boundaries of the optical grating, as shown inFIG. 4. However, accurate periodic sampling of the modulated lightproduced as light from the moving object passes through the opticalgrating is sufficient for making the velocity measurement, and the extracomplications of capturing and analyzing images is eliminated. In thepreferred approach, a lens 48 is used to collect the light transmittedby the optical grating and deliver it to a photodetector 50. It shouldbe noted that other optical elements, such as concave mirrors, can beused in place of the lenses discussed above.

In a preferred application, objects 18 are preferably biological cellsentrained in a fluid. The fluid is confined to a narrow column passingthrough FOV 38 of the optical system by hydrodynamic focusing with aflow sheath. The cells are kept within the depth of field of lenses 40and 44, assuring good focusing and, therefore, a modulation amplitudesufficient for the determination of the velocity of the object.

Light from a single object moving through the FOV at a uniform velocitywill, when modulated by the optical grating, have a frequency directlyproportional to the velocity, as defined by the following relation:$f = \frac{v}{s}$

where:

f=frequency (Hz)

s=grating pitch (microns)

v=velocity (microns/sec).

The amplitude of the signal generated at the photodetector by light froma single object will follow the contour of the illumination field. Ifthe illumination field profile has a Gaussian shape, for example, thesignal is described by the equation:

 x(t)=A ₀ e ^(−(t−t) ^(_(pk)) ⁾ ₂ ^(/τ) ₂ e ^(j2πf(t−t) ^(₀) ⁾ +A _(L)

where:

A₀=peak amplitude

A_(L)=leakage amplitude from stray light

t_(pk)=time of arrival at peak of illumination field

τ=envelope decay constant

t₀=time of arrival at edge of grating image.

FIG. 6 shows a typical embodiment of a photodetector signal conditioningand capture system. A central feature of this system is the use of abandpass filter 54 to filter the signal from the photodetector 50 (afterthe signal is amplified). The purpose of this bandpass filter is toreject a direct current (DC) component, A_(L), and to eliminate anyfrequencies above a Nyquist limit, f_(samp)/2, of an analog-to-digitalconverter (ADC) 56, where f_(samp) is the highest light modulationfrequency of interest. A variable-gain amplifier 52 is used to adjustthe amplitude of the signal to match the dynamic range of ADC 56. Thedigitized signal is delivered to a digital signal processor 58 foranalysis. Digital signal processor 58 can comprise a programmedcomputing device (e.g., a microprocessor and memory in which machineinstructions are stored that cause the microprocessor to appropriatelyprocess the signal), or an application specific integrated circuit(ASIC) chip that carries out the processing, or a digital oscilloscopethat includes such signal processing capability. FIG. 7A shows anexemplary unfiltered photodetector signal 60 generated by light from asingle object passing through the optical grating field, while FIG. 7Bshows an exemplary filtered photodetector signal 62, after applicationof bandpass filter 54 (see FIG. 6) to the signal.

As noted above, the present invention includes four distinct preferredembodiments. Those four preferred embodiments employ three differenttechniques for analyzing the signal from the photodetector, to deliveraccurate velocity estimates for the objects. The different signalprocessing methods are described in detail below.

As noted above, one preferred use of the velocity measurement in thepresent invention is to provide timing signals to optical systems thatdetermine characteristics of small moving objects, such as a flowcytometer. In non-imaging photomultiplier tube (PMF) instrumentscommonly known as flow cytometers, estimates of flow velocity are usedfor correcting measurements that depend on signal integration time andto accurately delay the sorting of a cell after its analysis. Theoptical grating-based velocity detection methods can be used to improvethe accuracy and reliability of such flow cytometric measurements andthe purity of sorted cell samples by providing a more accurate flowvelocity estimate.

The flow imaging systems disclosed in commonly assigned U.S. Pat. No.6,249,341, issued on Jun. 19, 2001, and entitled IMAGING AND ANALYZINGPARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS, as well as in commonlyassigned U.S. Pat. No. 6,211,955, issued on Apr. 3, 2001, also entitledIMAGING AND ANALYZING PARAMETERS OF SMALL MOVING OBJECTS SUCH AS CELLS,demand very accurate measurements of flow velocity for clocking the TDIdetector. The transfer of charge from one row to the next in the TDIdetector must be synchronized with the passage of objects through theflow cell. Note that the specification and drawings of each of these twopatents have been specifically incorporated herein by reference.

The theory of operation of a TDI detector, such as those employed in theabove-noted patent references is shown in FIG. 8. As objects travelthrough flow tube 16 and pass through the volume imaged by the TDIdetector, their images travel across the face of the TDI detector. TheTDI detector comprises a charge coupled device (CCD) array 64, which isspecially designed to allow charge to be transferred on each clock cyclein a row-by-row format, so that a given line of charge remains locked toor synchronized with a line in the image. The row of charge is clockedout of the array into a memory 66 when it reaches the bottom of thearray. The intensity of each line of the signal produced by the TDIdetector corresponding to an image of an object is integrated over timeas the image and corresponding signal propagate over the CCD array. Thistechnique greatly improves the SNR of the TDI detector compared tonon-integrating type detectors—a feature of great value when respondingto images from low-level fluorescence emission of an object, forexample.

The operation of the TDI detector can be understood from FIG. 8 byobserving the traversal of object 68 across the region imaged by CCDarray 64 and the history of the charge produced in response to the imageof object 68 by CCD array 64. The charge is transferred from row to rowin the array as the image of the object travels down the array. When arow of charge reaches the bottom of the array, it is transferred intoscrolling memory 66, where it can be displayed or analyzed. In FIG. 8,objects 70 and 72 traverse flow tube 16 ahead of object 68, while anobject 74 traverses flow tube 16 after object 68. Proper operation ofthe TDI detector requires that the charge signal be clocked down the CCDarray in synchronization with the rate at which the image of the objectmoves across the CCD array. An accurate clock signal to facilitate thissynchronization can be provided if the velocity of the object is known,and the present invention provides an accurate estimate of the objectsvelocity, and thus, the velocity of the image over the CCD array of theTDI detector.

FIG. 9 shows the integration of the velocity detector into a TDI-basedobject imaging system 10 e. The signal from photodetector 50 isprocessed by a signal processor 84, and may optionally carry outfunctions such as amplification and filtering. Additional details of thesignal processing are provided below. Preferably, signal processor 84comprises a programmable computing device, but an ASIC chip or digitaloscilloscopes can also be used for this purpose. The frequency of thephotodetector signal is measured and the velocity of object 18 a iscomputed as a function of that frequency. The velocity is periodicallydelivered to a TDI detector timing control 82 to adjust the clock rateof a TDI detector 80. The TDI detector clock rate must match thevelocity of the image of the object over the TDI detector to within asmall tolerance to minimize longitudinal image smearing in the outputsignal of the TDI detector. The velocity update rate must occurfrequently enough to keep the clock frequency within the tolerance bandas flow (object) velocity varies. Note that a dichroic beam splitter 76has been employed to divert a portion of light from object 18 a tophotodetector 50, and a portion of light from object 18 a to TDIdetector 80. An imaging lens 78 focuses an image of object 18 a onto TDIdetector 80.

FIG. 10 shows how a velocity detection system 88 is preferably employedby TDI detector timing control 82 (shown in FIG. 9). Note that velocitydetection system 88 can be configured as shown in FIG. 9, or provided inother configurations (such as shown in FIG. 5). Velocity detectionsystem 88 provides a clocking signal indicative of the velocity of aflow or of objects in a flow for synchronizing the movement of images ofthe objects over a TDI detector with the movement of charge responsiveto the images. The TDI detector is preferably included in a flowcytometry system, although other applications of the present inventionare contemplated. Preferably, the velocity detection system iscontrolled by a CPU 90, which executes a velocity detection supervisorprogram defined by machine instructions that are stored in memory (notseparately shown). Note that CPU 90 can also be employed to carry outthe signal processing function of velocity detection system 88. Theclocking of the charge through a TDI detector 94 is accomplished by avertical shift controller 92 and a horizontal readout controller 96,both of which are driven by a TDI detector timing control system 82. Thevelocity detection system 88 passes a clock frequency command to TDIdetector timing control system 86 to set a rate at which rows of chargeare shifted down the TDI detector array. Detector timing control system86 synchronizes horizontal readout controller 96 with vertical shiftcontroller 92.

The image information leaves TDI detector 94 as an analog signal, whichis then amplified with an amplifier 98 and digitized by an ADC 100. TheADC output is stored in a memory 102 under the control of timing controlsystem 86, where it can be accessed for display and analysis.

Another embodiment of a TDI-based flow imaging system 10 e is shown inFIG. 11. This embodiment is intended to address the problem ofsynchronizing the TDI detector to individual objects traveling atdifferent velocities, as may be the case in systems with poorhydrodynamic focusing. In TDI-based flow imaging system 10 f, thevelocity measurement is performed upstream of the point where imagecapture occurs. Velocity measurements are updated sufficiently rapidlyto be passed forward to the TDI detector timing controller in time forthe TDI detector clock (not separately shown) to be set to match thevelocity of an image of a particular object moving across the TDIdetector. The configuration of flow imaging system 10 f is similar tothat shown in FIG. 5, except that FOV 38 for velocity detection isseparate from a FOV 38 a used for TDI image acquisition. Imaging system10f uses a separate light source 12 a, separate lenses 36 a, 40 a, 44 aand a separate filter 42 a disposed in the collection path for lightfrom the objects that is directed to TDI detector 80. The photodetectorsignal is processed using a signal conditioning component 104 and a FFTbased fast velocity calculator 105 that is sufficiently fast to delivernew velocity estimates for objects to timing controller 82 in less timethan required for the objects to travel from velocity measuring FOV 38to imaging FOV 38 a. Note that in imaging system 10 f, signal processingblock 84 of FIG. 10 is separated into signal conditioning component 104and fast velocity calculator 106.

Accurate cell velocity measurements can also be employed to increasesort purity in droplet-based flow sorters. Such systems are typicallyPMT-based flow cytometers equipped to sort cells by their lightscattering characteristics or fluorescence emission. In such systems,the characterization of a cell is performed just as the liquid carryingthe cell leaves the droplet-forming nozzle. Based on an opticalmeasurement, the unbroken part of the stream is either charged or notcharged before the droplet containing the cell breaks free from thestream. An electrostatic field is used to deflect the charged dropletsinto a container separate from the container catching the unchargeddroplets.

FIG. 12 illustrates an instrument in which the optical grating-basedvelocity detection system of the present invention is used both tosynchronize a TDI detector for capturing images and for timing thedroplet charging system. In system 10 g, both velocity detection andimage capture are accomplished in common FOV 38. Images from TDIdetector 80 are delivered to a high-speed cell classifier 110. Theclassifier searches the images for objects of interest. Once such anobject has been found, the classifier automatically decides on the basisof the characteristics of that object whether the object should besorted into a container 130 or a container 132. If the decision is madeto place an object into container 132, the classifier triggers thecharging system, which comprises a time delay operator 112, a chargepulser 114, and a charge collar 116. The time delay used by time delayoperator 112 is set to match a transit time of an object from FOV 38 toan attached droplet 120, according to the velocity measurement performedin signal processing block 84 of the velocity detector. Note that asdescribed above, the velocity detector includes optical grating 46,photodetector 50, and signal processing block 84. Charged droplets 122are deflected into container 132 by the static electric field betweenelectrodes 124 and 126. The distance between the optical sensing regionand the charge collar is made long enough to provide sufficient time forimage-based object classification to reach completion. In this way, alarger and more complex set of features can be used in the sortingdecision than would be the case in conventional cell-sorting flowcytometers.

FDVM of Velocity of Objects on a Support

The first and second embodiments are directed to FDVM methods thatconvert light from cells or other objects into an amplitude modulated(AM) signal with a characteristic frequency that is proportional tovelocity. Any number of cells traveling at the same velocity, e.g., in afluid flow, can be in the sensitive region simultaneously, and themodulated light produced in response to the motion of each will have thesame fundamental frequency, differing only in phase. Unlike the priorart time-domain methodology, the FDVM method requires no synchronizationand is highly tolerant of variability in the fine structure of thetime-based waveform generated by the cells.

In the FDVM method, moving luminescent or illuminated cells are imagedonto a ruling of transparent and opaque bars to generate an amplitudemodulated light signal. The optical magnification and ruling pitch arechosen such that the bars are approximately the size (e.g., diameter) ofthe cell. The pitch of the ruling used in the optical grating isuniform. Therefore, the light collected from cells is alternatelyblocked and transmitted through the ruling as the cell traverses thesensitive region. The modulated light is directed toward a detector,producing an analog output signal with a fundamental frequencyproportional to the cell velocity. The analog signal is converted todigital samples. An FFT algorithm decomposes the resulting digitalsignal into spectral peaks in the frequency domain, which are processedto determine the velocity. A first FDVM embodiment is directed to amethod in which objects are deposited upon a support, and the support ismoved through the FOV. A second FDVM embodiment is directed to a methodin which objects are entrained in a fluid that is caused to flow throughthe FOV.

A block diagram of the first FDVM embodiment of a velocity detectionsystem is shown in FIG. 13. Beads are deposited on a slide 131 that isdriven through the FOV. For an initial feasibility study, the objectsemployed comprised 7.9 μm diameter beads (purchased from BangsCorporation) fixed to a moving microscope slide. Movement of the slidethrough the FOV was produced by mounting the slide on a closed-loop DCservo stage 133 (available from Newport Corporation), and the samplespeed was monitored using a one micron resolution linear encoderincluded in the stage. The stage had a maximum velocity of 100 mm/s,controlled using a proportional-integral-derivative (PID) control system(available from National Instruments Corporation). The linear encoderindependently monitored the movement of the slide (and hence, themovement of the beads on the slide) to provide comparative dataavailable to confirm the accuracy and precision of the FDVM velocitydetection system. While it is expected that determining the velocity ofobjects entrained in a fluid will have widespread application, it isalso anticipated that the moving support (slide) embodiment will also beuseful, particularly for objects that cannot be easily or stablyentrained or suspended in a fluid.

The sample beads were illuminated with light from a diode laser 134(from Thor Labs, Inc.) so that light striking the beads was scatteredinto the optical collection system. The moving sample image wasprojected at approximately 5× magnification by an objective 136 and alens 138, onto an optical grating 135 having a ruling of 50 micron bars(available from Gage Technologies), oriented at right angles to themotion of the sample. The ruling and sample were then imaged together onan adjustable slit 139 by an imaging lens 137, disposed to simulate thefield of view of a flow-based instrument. The light passing through theslit was then collected by a lens 141 and directed onto a PMT 148(Hamamatsu Corp., Model 5783-01) such that the aperture of the opticalsystem was imaged onto the PMT (143). In this manner, there was nomovement of the signal across the PMT as the bead images traversed theruling.

The signal processing portion of this embodiment of a velocity detectionsystem is also depicted in FIG. 13. The signal from the PMT wasamplified and highpass filtered through an amplifier/filter 150(Stanford Research, Model SR570). The filtered signal was then digitizedusing an analog-to-digital converter 147, and the digitized signal wasprocessed using an FFT processor 149 to produce a frequency spectrumwith a well-defined peak at the frequency corresponding to the velocityof beads. The FFT spectrum was smoothed using a moving average filter151 and the zero crossing of the derivative of the smoothed FFT spectrumwas determined in processing blocks 153, 155, and 157. All signalprocessing was performed on a digital storage oscilloscope (from LeCroyCorp.). The velocity of the objects on the slide was then calculated bytaking the product of frequency defined by the zero crossing, the rulingspacing, and the inverse of the magnification in a velocity conversionblock 159. The precision of the measurement was enhanced by linearlyinterpolating between the derivative data points to better define thezero crossing frequency.

FIG. 14A shows an image of the beads, ruling, and adjustable slit. Theimage was acquired by inserting a beam splitter, lens and detector afterthe ruling, placing a light source behind the slide, and opening theslit for clarity. The beads were magnified 4.92× before being imaged onthe ruling, which had a line width of 50.8 μm (9.842 Ip/mm). FIG. 14B isa scattered light image in which dark field illumination was employed,and the slit closed down to 200 microns (40 microns in object space), asit was during data acquisition. In operation, the motorized stage movedthe beads across the field of view at 20 mm/s (left to right in theillustrated example), while data was acquired for approximately onesecond.

Using the methods and apparatus discussed above, data were taken inthree experiments to determine the precision and accuracy of the thistechnique. FIGS. 15, 16, and 17 summarize the results of theseexperiments. In the results of the precision experiment shown in FIG.15, the stage was commanded to move at 20 mm/s in 34 separate runs. Thevelocities measured by the encoder on the stage and by the FDVM methodof the present invention were both recorded and plotted. To calibratethe FDVM method, a correction constant was determined by taking thequotient of the first commanded velocity and the frequency peak producedby the FDVM method. Each subsequent measurement was multiplied by thisvalue. The precision of the FDVM method was determined by calculating acoefficient of variation (CV) for 34 separate runs. By this measure, theprecision of the encoder method is 0.09% and the precision of the FDVMmethod of the present invention is 0.01%, as shown in FIG. 16. Thisexperiment demonstrates that the precision of the FDVM method exceedstargeted performance requirements by a factor of fifty.

It should be noted that the poorer apparent performance of the encodermethod is likely the result of the servo feedback system's internalvelocity calculation. Rather than making one velocity measurement perrun using all 20,000 counts, the servo system makes a velocitymeasurement every 60 counts for the purposes of real-time motioncontrol. The stage feedback system supplied a function to average theindividual velocity measurements within a run. Each point in the encoderprecision plot is therefore the average of 333 individual velocitymeasurements.

The results of the linearity experiment are shown in FIG. 17. The stagewas commanded to move over a velocity range from 5 mm/s to 100 mm/s asspecified in the performance requirements. Velocity measurements weretaken using the FDVM method and the stage encoder. Over this range bothmeasurements produced highly correlated R² values of unity with slopesof 1.0002 and 1.0007 for the FDVM method and stage encoder,respectively. These results demonstrate that the FDVM method of thepresent invention has good linearity over a range of velocitymeasurements exceeding an order of magnitude.

FIG. 18 is an image captured using a TDI detector configured to view thesample slide, as in FIG. 2. The TDI detector or detector capturesunblurred imaged of objects, which move at the same rate as the chargethat is clocked across the chip during imaging. Because the TDI detectoris located behind the stationary ruling, the ruling has blurred acrossthe entire field of view. The ruling is responsible for some imagedegradation as each bead image traverses the entire ruling during theimaging process. The TDI detector's pixel clock was generated using thevelocity determined by the FDVM method of the present invention.Although a comprehensive analysis of the image has not been performed,it is apparent that the velocity accuracy is sufficient to prevent imageelongation in the horizontal axis of motion of the TDI detector.

The goals of the feasibility study employing beads on a slide were todevelop a velocity detection system with high precision, high accuracy,good linearity and tolerance of wide variations in sample density. Thesegoals were exceeded and the system was successfully used to captureimages using a TDI detector. The 0.5% feasibility requirements were setto ensure less than one pixel blur when using the velocity detectionsystem in concert with a 100 stage TDI detector. In fact, thefeasibility system demonstrated precision, accuracy, and linearitybetter than 0.05% and therefore can be used with a 1000 stage TDIdetector. In the context of the cell analysis system being developed inwhich the present invention will be included, more stages enable theimage to be collected over a larger field of view, thereby increasingthe integration time and the sensitivity of the instrument. Conversely,if the field of view is held constant, the pixel size can be reduced,increasing the spatial resolution of the instrument. Accurate velocitydetection is also beneficial for cell sorting, where knowledge of thestream velocity can be used to actively adjust drop delays to compensatefor drift over the course of an experiment.

Supervisory Control of Velocity Measurement Systems

In all embodiments, the present invention entails the steps of (1)formation of images of the objects of interest focused in the plane ofthe optical grating, (2) delivery of the modulated light transmitted bythe optical grating to the surface of a photosensitive detector, (3)conversion of the modulated light signal to an electronic signal, (4)signal acquisition and processing, and (5) velocity determination.Preferably one or more of these operations will be brought under thecontrol of supervisory software by interfacing the velocity measurementsystem with a general purpose computer or other computing device.

FIG. 19 is a functional block diagram of a signal acquisition andanalysis system controlled by a supervisor program 142, preferablyexecuted by a CPU 144 of a programmable computer. Alternatively,supervisor program 142 can be executed by a corresponding CPU in adigital oscilloscope, or by an ASIC chip. A signal from photodetector 50is processed via signal acquisition and conditioning process block 104,velocity computation process block 105 (note that fast velocitycomputation process block 106 of FIG. 11 employs FFT processing, while,velocity computation process block 105 is more generalized, and canemploy other types of signal processing, as opposed to just FFT signalprocessing), and a velocity running average computation block 140. Basedon the velocity of an object that was determined, supervisor program 142provides a clocking signal to a timing generator 146 that controls TDIdetector 80. Note that the TDI detector is only one device exemplarydevice that can employ the present invention. It is expected that othertypes of devices can be provided a timing signal in this manner.

FDVM of Objects in Flow

In the second preferred embodiment of the present invention, the lightfrom the objects is modulated by the optical grating, and the modulatedlight is sensed by the photodetector shown in FIG. 5. The functionalblocks used to capture, process, and analyze the photodetector signalare shown in FIG. 20. Details of a multistage digital signal processingoperation 150 for processing the incoming signal are illustrated in FIG.21. The entire system, shown in FIG. 20, operates as a pipelineprocessor in which blocks of signal samples, and farther down thepipeline, parameters calculated from the blocks of samples are passedfrom one operation to the next in an uninterrupted sequence.

As explained above in connection with FIG. 6, the signal produced byphotodetector 50 is input to variable gain amplifier 52. The output ofvariable gain amplifier 52 is filtered by bandpass filter 54 to removethe DC bias caused by stray light and by bias voltage of variable gainamplifier 52 and to eliminate frequencies above the Nyquist limit of ADC56, this limit being equal to one-half of the sample frequency,f_(samp). After bandpass filtering, the signal swings in both thepositive and the negative direction around a mean value of zero.

ADC 56 samples the signal at frequency f_(samp) and encodes the signalinto a series of digital samples representing the signal amplitude ateach sequential sample time. The converter must retain the bipolarnature of the signal by encoding the signal into a number system such asthe 2's complement format, which accommodates both positive and negativenumbers.

As an alternative, ADC 56 could be placed immediately after variablegain amplifier 52 and bandpass filter 54 could be implemented as adigital instead of an analog type filter. The signal applied to the ADCwould then be unipolar, and the signal would be encoded into a simplebinary number for processing.

In a multistage digital signal processing block 150, one signal segmentis processed. Referring now to FIG. 21, in block 150, a sequentialseries of signal samples of predetermined length, N_segment, is analyzedto extract the mean frequency of the photodetector signal and to convertthat frequency to an estimate of the object velocity. The first step 160of this operation is to capture the desired number of samples for thesegment from the incoming signal.

Optionally, the next signal processing step 162 applies an amplitudewindowing or apodization function to the signal segment that was justcaptured. Without this apodization step, the abrupt truncation of thesignal at the ends of the segment would spread the frequency componentsin the signal over a collection of sidebands, the frequency andamplitude of which conform to the Fourier Transform of a rectangularwindow, a sine function in frequency space. Those skilled in the artwill recognize that apodization by a function such as the Hamming,Hanning, or raised cosine functions, for example, will substitute asmoother sideband structure of lower amplitude in place of the sinefunction. The reduced sideband amplitude improves the accuracy ofestimating the mean frequency of the photodetector signal, especially inthe presence of velocity dispersion. Alternatively, apodization can beperformed optically by illuminating the FOV using a smooth-shoulderedintensity profile, thereby eliminating the abrupt truncation of thesignal at the edges of the FOV. In still another method of apodization,the ruling may be superimposed on a varying transmission gradientfilter, which smoothly attenuates the optical signal at the edges of theFOV.

The optional apodization operation in step 162 is followed by executionof a complex FFT function in a block 164. The complex FFT algorithm isutilized by applying the signal as the real input to the FFT andapplying an array of length N_segment with all values set to zero as theimaginary input to the FFT. Alternatives exist for utilizing the FFTalgorithm more efficiently for real-number transforms, but those methodsinvolve packing the input arrays in special patterns and unpacking theoutput arrays. Such methods can be used, however, to save processingtime.

The resulting complex number spectrum is then applied to an operator ina block 166 that converts the real and imaginary parts of the spectrumto the magnitude of the spectrum using the following relation:

M _(j) ={square root over (Re_(j) ²+Im_(j) ²)}

where:

M_(j)=magnitude at sample j

Re_(j)=real part of sample j

Im_(j)=imaginary part of sample j.

Typically, this operation will be implemented using a look-up table orfast approximation algorithm to speed execution.

Typically, the velocity of the objects to be imaged with a TDI detectorwill deviate very little from the mean velocity. Consequently, the powerin the spectrum of the signal from the photodetector will beconcentrated in a narrow band around a mean frequency, and the spectrumoutside this band can be ignored in the computation of mean frequency.

FIG. 22 is the spectrum produced by a sinusoidal burst with centerfrequency 2500 Hz and a Gaussian-shaped envelope. A single objectpassing through the flow cell of a flow imaging system such as thatillustrated in FIG. 9 might produce such a signal. A simple peakdetector is applied to the spectrum in FIG. 22 in a block 168 of FIG.21, to localize a region 178 of the spectrum of interest for analysis.FIG. 23 shows a segment 180 of the spectrum centered on the peak of thespectrum of the signal burst. This segment contains nearly all of thepower in the spectrum and can be utilized for computing the meanvelocity.

In a block 172 of FIG. 21, the mean velocity is determined by findingthe mean frequency on the scale of FFT bins from the signal segment ofFIG. 17. The following relation describes this calculation:$\overset{\_}{n} = \frac{\sum\limits_{n = a}^{b}\quad {{nS}(n)}}{\sum\limits_{n = a}^{b}\quad {S(n)}}$

where:

a, b=endpoints of sample in window

S=magnitude of spectrum

{overscore (n)}=mean FFT bin (floating point).

The mean frequency in Hz is computed from the mean FFT bin number asfollows:

{overscore (f)}(Hz)=2·{overscore (n)}· f _(Nyq) /N

where:

f_(Nyq)=Nyquist frequency

N=FFT length

{overscore (f)}=mean frequency.

Finally, a mean velocity 176 is found by multiplying the mean frequencyby the optical grating pitch:

{overscore (v)}={overscore (f)}·s

where:

s=grating pitch (microns)

{overscore (v)}=velocity (microns/sec).

The velocity detection system must accommodate the possibility that verylittle or no signal was captured in the signal segment being processed.In the present embodiment of the invention, the magnitude of thespectrum integrated over the local region around the peak of thespectrum is computed in a block 170, as follows:$M_{sig} = {\sum\limits_{n = a}^{b}\quad {S(n)}}$

where:

M_(sig)=integrated magnitude, this segment

S=magnitude of spectrum

a=first bin of local region around peak

b=last bin of local region around peak.

As will be appreciated from the description of the supervisor programfor the velocity detection system that follows, a running record of themean velocity will be maintained by the supervisor and used to establishthe boundaries, a and b, of the local region for computing the meanfrequency and an integrated magnitude 174.

Referring back to FIG. 20, the mean velocity and integrated signalmagnitude from operation 150 are applied to a decision step 152. Thedecision step 152 yields two possible outcomes: (1) the SNR of thesegment being processed is adequate for computing the velocity, so thatthe new velocity value is added to a velocity list 154, or (2) the SNRis inadequate (below a predefined value) for computing the velocity, andthe velocity list remains unchanged. If a new velocity value is added tothe velocity list, the oldest value on the list is deleted (once thelist is full). Velocity list 154 and a running average calculation in astep 156 deliver a new velocity estimate every time a new signal segmentis processed. The running average velocity is the average over apredetermined number of velocity values, m. The number of values, m,used in the running average can be increased to improve the accuracy ofthe running average or decreased to improve the responsiveness of thevelocity detector to rapid changes in velocity.

The velocity detector must adapt to variations in flow velocity andphotodetector SNR in order to produce accurate and reliable velocityestimates. Supervisor program 142, shown in FIG. 19, is used to controlthe velocity detector and to coordinate the operations of the velocitydetector with those of the rest of the imaging system. FIG. 24 is aflowchart showing the steps implemented by the supervisor program forthe second embodiment of the present invention. The program's threeprincipal outputs, the SNR decision threshold, the spectrum integrationlimits, and the photodetector amplifier gain, are fed back to thevelocity measurement system to optimize its performance.

Operation of the velocity detection system is initiated with aninstrument calibration step 190, in which the noise from thephotodetector channel is determined and analyzed in the absence of anoptical signal. This can be accomplished by turning off the lightsources in the system or stopping the flow of objects through the flowcell. The purpose of the noise measurement is to establish a referenceagainst which the information-bearing signal will be compared forsetting a threshold for accepting or rejecting velocity measurements.

The calibration operation measures the noise level at a plurality ofamplifier gain settings and stores these measurements in a table 192 ofnoise level vs. gain. Table 192 is used to select an initial gainsetting for amplifier 52. As the amplifier gain is varied to regulatethe signal strength during normal operation, the correct noise level forsetting the decision threshold is read from table 192 and applied tothreshold calculator 194. Once the calibration operation has beencompleted, the light source or sources are turned on, and objects areintroduced into the flow stream for image acquisition, as shown in astep 196.

The next task of the supervisor program is to search for the peak in thespectrum in a step 198 and set the upper and lower boundaries of thespectral region to be analyzed. In the absence of any a priori knowledgeof the flow speed, this initial search must span the entire range offrequencies in the spectrum, and may entail capturing a number of signalsegments until a strong peak representing a spectral peak frequency 200is found. The location of that peak will be used to set the local regionboundaries, using knowledge of the expected width of the spectrum.

This width is a function of the beam profile of the illumination field,the shape of the apodization function, and the predicted variance ofobject velocities. This information will be understood from the designof the instrument.

With the photodetector amplifier gain set to a starting value and thedecision threshold and integration limits established, pipelineprocessing of signal segments commences. Each time a segment isprocessed, running average velocity value 158 is added to a list 204 andthe oldest value in the list is deleted. The velocity values in list 204are then averaged in a step 206. This long-time average of the velocityis used in a step 202 in which the boundaries of the local spectralregion to be analyzed are set. The process of regulating the integrationlimits constitutes a feedback control loop in the supervisor program.The response time of this loop can be modified by adjusting the numberof samples maintained in list 204 and averaged in step 206.

The gain of the photodetector amplifier is regulated during systemoperation as well, in order to optimize the SNR of the velocity detectoras specimen characteristics change. The amplifier gain regulation systemof the supervisor program in a step 208 provides for creating thehistogram of each signal segment, counting the number of samplesoccupying a predetermined number of levels near the top of theanalog-to-digital converter output scale in a step 210, maintaining alist of the most recent count results in table in a step 212, andanalyzing that table to generate a gain adjustment in a step 214.

The time of arrival of objects in the FOV of the velocity measurementsystem is a random variable. If the specimen contains a highconcentration of objects, the probability that an object will passthrough the FOV in a given time interval is high, and the count table ofstep 212 will contain many samples useful in setting the amplifier gain.However, if the specimen contains a very low concentration of objects,many of the signal segments processed by the velocity detection systemwill be devoid of signal, and the count values stored in the count tableof step 212 for those segments will be zero. Those skilled in the art ofautomatic gain control systems will recognize this problem as similar tothat of regulating the gain in radio receivers or studio microphoneamplifiers, in which the signal being processed may vary widely inamplitude and be interrupted. The common practice in such cases is touse a “fast attack,” “slow recovery” feedback control system. In such asystem, the sudden arrival of a high-amplitude signal will be met with afast reduction of amplifier gain to prevent saturation. On the otherhand, a prolonged interruption of the signal is met with a slow increasein gain, on the premise that a large signal is likely to arrive soon,but a persistent loss of amplitude, requiring higher gain may haveoccurred. The gain adjustment determined in step 214 will use a “fastattack,” “slow recovery” algorithm to regulate the photodetectoramplifier gain. Determination of running average 158, and steps 208 and210, and their associated feedback control mechanisms will sustainsequential processing of signal segments until terminated by thesupervisor program.

The new gain setting is set in a step 216 for variable gain amplifier 52in regard to noise calibration table 192. The response time of the gaincontrol feedback loop can be modified by adjusting the number of samplesmaintained in table 212 and analyzed in step 214.

TDVM of Objects using a Single Uniform Pitch Optical Grating

In the third preferred embodiment of the present invention, the lightcollected for velocity measurement is modulated by an optical gratinghaving a substantially uniform pitch and sensed by the photodetectorshown in FIG. 5, just as in the second embodiment. The analysis of thephotodetector signal, however, is performed in the baseband domain.Baseband demodulation is frequently used in communications and othersignal processing applications for receiver tuning and carrierrejection. The fundamental architecture of a baseband demodulator withthe additional capability of splitting the signal into upper and lowersidebands, is shown in FIG. 25 and is referred to herein as “thedouble-sideband receiver.”

As shown in FIG. 25, an incoming signal from the photodetector isapplied to multipliers (or mixers) 222 and 226, which multiply thesignal by two continuous sinusoidal wave functions, called localoscillator signals. The two local oscillator signals are at the samefrequency, but shifted in phase ninety degrees relative to one another.That is, a first local oscillator signal 224, which is the in-phaselocal oscillator signal, is a cosine function, while a second firstlocal oscillator signal 228, which is the quadrature-phase localoscillator signal, is a sine function. The mixers are followed bylowpass filters 230 and 232, which complete the baseband demodulation.

The effect of multiplying the signal from the photodetector by asinusoid of frequency f_(LO) is to offset the spectrum of the incomingsignal upward by f_(Lo), to create sum frequencies, and downward by−f_(LO), to create difference frequencies. In FIG. 26, a sinusoidalburst with the spectrum shown in a graph 250 at a center frequency of1500 Hz is applied to mixers 252 and 256, driven by local oscillatorsignals 254 and 258, set to 2000 Hz. At the output of the mixers, thecenter frequencies in the spectrum are the difference frequency, 500 Hz,and the sum frequency, 3500 Hz, as shown in graph 264. Lowpass filters260 and 262 suppress the sum frequencies, leaving only the differencefrequencies in the I(t 268 and Q(t) 266 signals, as shown in a graph270.

The lowpass filter outputs are the I and Q signals, i.e., the in-phaseand quadrature-phase signals. FIG. 27 shows how signals entering andleaving the baseband demodulator might look on an oscilloscope. Eachtime sample is a complex number representation of the input signal 272,with the I channel 268 representing the real part of the complex signal,and the Q channel 266 representing the imaginary part of the complexsignal. Graph 276 shows the in-phase output signal, while graph 274shows the quadrature-phase output signal. The I,Q pair conveys both themagnitude of the signal and the phase of the input signal 272 relativeto the local oscillators. A time series of I,Q pairs can represent bothpositive and negative frequencies, which derive from frequencies abovethe local oscillator frequency and below the local oscillator frequency,respectively. The time series of I,Q pairs is often referred to as the“analytical signal.”

The magnitude and the phase of the input signal can be calculated fromthe analytical signal using the vector operations shown in FIG. 28. Themagnitude of a signal 282 is computed in a step 278. I(t) and Q(t) arethe Cartesian projections of the vector M(t), therefore M(t) is just thelength of the hypotenuse of a right triangle with the other two sidesbeing I(t) and Q(t). The equation for calculating M(t), then, is:

M(t)={square root over (I(t)²+Q(t)²)}.

Accordingly, the angle between the real, I(t), axis and the hypotenuse,M(t), is the inverse tangent of Q(t)/I(t), or:

Φ(t)=arctan[Q(t)/I(t)].

The analytical signal offers a versatile method for tracking thephotodetector signal frequency in the velocity detector. The frequencyat every sample time is found by taking the time derivative of the phaseof the analytical signal in a step 280. However, as seen in a graph 284of Φ(t), the phase is a periodic function. The values π and −π definethe same angle, where the abrupt transitions occur in graph 284.

In order to calculate a phase derivative for each time sample, theperiodic Φ(t) function must be converted to a continuous function. FIG.29 illustrates the unwrapping of the phase of a constant frequencysignal. The function Φ(t) is shown in a graph 288. A polar plot 286shows a rotating vector representation of a constant amplitude, constantfrequency signal. It can be seen from plot 286 that the phase will makean abrupt transition from π to −π once per period. A phase unwrappingalgorithm 290 senses these transitions and corrects for them to producea monotonically increasing function of phase, as shown in a graph 292.

FIG. 30 describes the phase unwrapping algorithm. In a first step 294,the change of phase from one time sample to the next is computed withoutregard to the values of Φ(n) and Φ(n−1). In the next two stages, steps296 and 298, the presence of a transition across the π,−π boundary issensed and the phase derivative is corrected. The first part of bothsteps 296 and 298 is to detect that the phase has moved from onehalf-plane to the other half-plane. If Φ(n−1) was zero or positive, thenthe transition was from the upper half-plane to the lower half-plane,and the step 296 is executed. If Φ(n−1) is negative, then the transitionwas from the lower half-plane to the upper half-plane, and the step 298is performed. In step 296, a further test is performed to determine ifΦ(t) has changed in the negative direction by more than π. If so, thenthe transition between half-planes was at the π,−π boundary. In thiscase, an offset of +2π is applied to ΔΦ, which eliminates the impact ofthe boundary transition. In a similar manner, if step 298 detects thatthe phase has rotated across π,−π boundary in the clockwise direction,it subtracts 2π from ΔΦ to eliminate the impact of the boundarytransition. Application of the unwrap algorithm to the phase signal fromthe baseband demodulator is used to generate a smooth phase plot,Φ_(m)(t), as shown in graph 292 of FIG. 29. The slope of this plot isthe radial frequency, ω(t).

Any signal generated by a physical system will contain some randomnoise. Because ω(t) is computed using a difference operator, and truerandom noise is uncorrelated from sample to sample, the accuracy of theω(t) calculation will degrade rapidly with decreasing SNR. For thisreason, time samples of ω(t) are accepted into the velocity computationonly if the magnitude of the signal is above a predefined threshold.This concept is illustrated by FIGS. 31A and 31B. In a graph 302 of FIG.31A, a threshold of 0.1 is applied to the magnitude, M(t). The basebandfrequency is computed only for those samples exceeding the threshold,yielding a result like that seen in a graph 308 of FIG. 31B. The term“fractional frequency” 310 used in graph 308 means the frequencyexpressed as a fraction of the Nyquist limit in baseband. The fractionalfrequency can have a value in the range from 0.0 to 1.0.

FIG. 32 shows the signal processing and data pathways for the thirdembodiment. The signal from photodetector 50 is applied to variable gainamplifier 52, the gain of which is regulated by the supervisor programto optimize SNR. The amplified signal is applied to bandpass filter 54to remove DC offset and to limit the signal bandwidth to preventaliasing. The filtered signal is converted to a sequence of digitalsample by ADC 56. Mixers 222 and 226 and lowpass filters 230 and 232(FIG. 25) are implemented in the baseband conversion of a step 311. Thebaseband signal pair I(n), Q(n) is used for two steps. The first is thegeneration of the upper sideband and lower sideband signals 314 and 322in a step 312. The second is the measurement of the velocity of objectspassing through the flow cell.

FIG. 33 illustrates the generation of the upper and lower sidebandsignals from the I,Q signal pair. An I(n) signal 326 and a Q(n) signal328 are each processed by the Hilbert Transform operator, applied insteps 234 and 236. Those skilled in the art will recognize that thisoperator delays the input signal by π/2 radians (90 degrees). Note thatthe phase rotation is not a time delay, because the rotation is π/2,independent of the frequency of the incoming signal, over a broad rangeof frequencies. However, a time delay is inherent in the HilbertTransform algorithm, and must be matched by time delays 238 and 240. Thefinal stage of the sideband separation is that of summing at node 242the rotated Q(n) signal with the unrotated I(n) signal to generate anupper sideband signal, USB(n) 330, and of summing at node 244 therotated I(n) signal with the unrotated Q(n) signal to generate a lowersideband signal, LSB(n) 332. Summation at node 242 cancels signalvectors rotating counterclockwise in I,Q plane 286 (see FIG. 29) andreinforces those rotating clockwise in the I,Q plane. Summation at node244 cancels signal vectors rotating clockwise in I,Q plane 286 andreinforces those rotating counterclockwise in the I,Q plane. The upperand lower sideband signals are used by the supervisor program duringsystem start-up to search for the photodetector frequency and to set thelocal oscillator frequency for the baseband demodulator.

The I,Q complex signal is also applied to the pipeline process in ablock 316 (see FIG. 32), which detects and segments signals fromindividual objects in the flow stream, tests these signals againstpredetermined acceptance criteria, and computes the object velocity fromthe accepted signals. The details of the steps implemented in block 316are shown in FIG. 34.

The signal threshold concept illustrated in graph 302 (FIG. 31A) is usedto segment the signal stream into sample packets, each of whichrepresents an object or an aggregate of objects in the flow stream. Themost accurate velocity measurements are those derived from the signalsfrom isolated single objects. Signals from aggregate objects, i.e.,signals from multiple objects coexisting in the FOV of the velocitydetector, carry phase errors caused by the interference among thesignals from the individual objects. The segmentation then accepts onlythose signals with an envelope width close to that predicted for asingle object passing through the FOV at the current expected velocity.Because the envelope width is inversely proportional to velocity, thesupervisor program tracks the known velocity and corrects the widthlimits as the velocity changes.

Referring to FIG. 34, each base pair of the I,Q complex signal isanalyzed starting in a block 334. Segmentation of a packet begins whenthe magnitude of the signal crosses a threshold 371 while rising,detected in an step 336, and ends with the magnitude falls back acrossthreshold 371, detected in a step 342. The sample count, n, and theunwrapped phase, Φ_(m)(n), are set to zero in a step 338 when the risingedge of the packet is detected. The unwrapped phase is computed in astep 344 for each sample following the rising edge, and the sample countis incremented each time a new sample is acquired in a step 340. Oncethe falling edge of the packet is detected, the sample count, n, istaken as a width 364 of the packet. The phase samples Φ_(m)(0) throughΦ_(m)(n) are used in the computation of the average frequency of thesignal packet, and, subsequently, the velocity of the object.

However, each packet must meet two criteria before being accepted as auseful signal. First, the packet width is compared to upper and lowerwidth limits 366 in a step 346. The packet is rejected if the widthfalls outside those limits. The radial fractional frequency, ω(n), iscomputed for each sample within the packet in a step 348. The unwrappingalgorithm cannot deliver values outside of the range from −π to π, sincethe Nyquist limits are −π and π radians/sample. Division by π inoperation 348 expresses ω(n) as the dimensionless fraction of theNyquist limit. The variance is computed for the ensemble of values ω(1)through ω(n) in a step 350 and compared with a maximum limit 373 in astep 360. This limit is a constant determined empirically for deliveryof the required accuracy in the velocity measurements while limiting thenumber of rejected objects.

If the wave packet is accepted as representing a single object and ashaving an acceptably low frequency variance, an object velocity 368 iscomputed in a step 362 as follows:${\overset{\_}{f}}_{bb} = {\frac{\sum\limits_{i = 1}^{n}\quad {\omega (i)}}{n} \cdot f_{Nyq}}$

and

v _(o)(mm/sec)=({overscore (f)} _(bb) +f _(LO))·s

where:

ω(i)=fractional frequency for sample i

f_(Nyq)=Nyquist frequency for baseband

{overscore (f)}_(bb)=mean baseband frequency (Hz)

f_(LO)=local oscillator frequency (Hz)

s=grating pitch (mm)

v_(o)=particle velocity (mm/sec).

In FIG. 35A, a graph 370 represents a plot of the baseband frequencyversus time for the magnitude signal shown in graph 302 (FIG. 31A), butwith only the qualified signals retained. A graph 372 in FIG. 35B showsthe series of object velocities computed in operation 362 (see FIG. 34)from the baseband frequency data shown in graph 370.

For each accepted object, a velocity, v_(o), is delivered to a scrollingobject velocity list 318 of FIG. 32. Every time a new velocity value isadded to the scrolling list, the oldest value is removed from the list.A running average computation in a step 320 constantly determines arunning average 324 of the values in the scrolling velocity list at arepetition rate determined by the supervisor program.

FIG. 36 shows the structure of the supervisor program for the thirdembodiment of the present invention. Operation of the velocity detectionsystem is initiated with the instrument calibration in a step 374, inwhich the noise from the photodetector channel is determined andanalyzed in the absence of an optical signal. This step can beaccomplished by turning off the light sources in the system or stoppingthe flow of objects through the flow tube. The purpose of the noisemeasurement is to establish a reference against which the informationbearing signal will be compared for setting a threshold for accepting orrejecting phase samples.

The calibration operation measures the noise level at a plurality ofamplifier gain settings and stores these measurements in a table 376 ofnoise level vs. gain. Table 376 will be used to select an initial gainsetting for variable gain amplifier 52. As the amplifier gain is variedto regulate the signal strength during normal operation, the correctnoise level for setting the decision threshold will be read from table376 and applied to a threshold calculation in a step 378. Once thecalibration operation has been completed, the light source or sourcesare turned on, and objects are introduced into the flow stream for imageacquisition, as shown in a step 380.

The next task of the supervisor program shown in a step 382, is tosearch the spectrum for the photodetector signal. In the absence of anya priori knowledge of the flow speed, this initial search must span theentire range of frequencies in the spectrum, and may entail sweeping thespectrum a number of times until a strong signal is found. Step 382sweeps the frequency of the local oscillator and captures a short timesegment of upper sideband 330 and lower sideband 332 signals (see FIG.33). As the local oscillator is swept across the actual frequency of thephotodetector signal, the lower sideband amplitude will increase andthen drop. Then the upper sideband amplitude will increase and thendrop. For the broad sweep to locate the approximate photodetectorfrequency, the local oscillator is varied in large increments to speedthe search, and the search in step 382 measures the root mean square(rms) sum of the sidebands as follows:$P_{ush} = {\sum\limits_{i = 1}^{N}\quad {U^{2}\lbrack i\rbrack}}$$P_{isb} = {\sum\limits_{i = 1}^{N}\quad {L^{2}\lbrack i\rbrack}}$

 P _(sum) ={square root over (P_(usb) ²+P_(lsb) ²)}

where:

U[i]=upper sideband amplitude of ith sample

L[i]=lower sideband amplitude of ith sample

N=number of time samples in tested signal segment

P_(sum)=integrated sideband power for signal segment.

FIG. 37 is a graph 406 of the integrated sideband power versus the localoscillator frequency for the broad search sweep. The width of a powerenvelope 408 is two times the bandwidth of lowpass filters 230 and 232(see FIG. 25) in the baseband demodulator. The desired local oscillatorfrequency is located at a dip 410 between the two peaks in the powerenvelope. However, this frequency is poorly resolved because of thelarge steps used in search sweep 382.

Referring back to FIG. 36, a more accurate estimate of the desired localoscillator frequency is made in a step 384 by varying the localoscillator frequency over a narrow range covering the power envelope.This narrow sweep is illustrated by a graph 414 in FIG. 38, which is anoverlay of an upper sideband power 416, called P_(usb), and a lowersideband power 418, called P_(lsb), as a function of local oscillatorfrequency. The search in step 384 of FIG. 36 finds a frequency 420 inFIG. 38 at which the upper sideband and lower sideband are of equalpower. As will be evident in FIG. 38, this frequency is approximately2500 Hz. Under this condition, the local oscillator frequency isapproximately equal to the photodetector signal center frequency, andthe baseband demodulation system can be used to measure the exactphotodetector signal frequency.

With the magnitude threshold and the local oscillator frequency set,object processing can commence. During object processing, the supervisorprogram continuously monitors the sideband signals in a step 388 (FIG.36) using sample locations accepted by signal processing step 316 (seeFIG. 32). The selected sideband samples are used to monitor the balancebetween the power in the upper sideband signal and that in the lowersideband signal. Imbalance between the two sideband signals indicatesthat the photodetector frequency has shifted and that the localoscillator frequency should be adjusted. The sideband balance will berepeatedly computed in a step 388, the balance values stored in a table390, adjustments to the local oscillator will be computed in a step 394,and applied in a step 386. The number of values maintained in table 390can be modified to adjust the response time and stability of the localoscillator feedback loop.

The sideband signal levels are checked to determine if they have beenlost in a step 392. If both the upper sideband and lower sidebandsignals are lost, the supervisor program interrupts signal processingand returns to search routine 382 to tune the system back to thephotodetector signal frequency, if possible. The supervisor program willremain in the search mode until a signal is acquired or the velocitydetection system is turned off.

The gain of the variable gain amplifier is regulated during systemoperation as well, in order to optimize the SNR of the velocity detectoras specimen characteristics change. The amplifier gain regulation systemof the supervisor program implemented in a step 396 creates thehistogram of the peak magnitudes of the accepted signal packets. A step398 provides for counting a number of samples occupying a predeterminednumber of levels near the top of the analog-to-digital converter outputscale, and maintains a list of the most recent count results in a table400. That table is analyzed to determine a gain adjustment in a step402. The gain adjustment implemented in step 402 will use a “fastattack,” “slow recovery” algorithm, as described above, to regulate thegain of the variable gain amplifier.

The new gain setting is set in a step 404 and is provided to a noisecalibration table 376. The response time and stability of the gaincontrol feedback loop can be modified by adjusting the number of samplesmaintained in table 400 and analyzed in step 402.

TDVM of Objects Using Paired Non-uniform Optical Gratings

In the fourth preferred embodiment of the present invention, the lightcollected for velocity measurement is modulated by two optical gratingsand sensed by two photodetectors, as shown in FIG. 43. The velocity ismeasured by cross-correlating the signal from the first photodetectorwith that from the second photodetector, yielding a time-of-flight valuethat is converted into a velocity of the object.

The cross-correlation of two signals is carried out by convolving thetwo signals and extracting information from the output of theconvolution operation, which is called the correlogram. The convolutionin the time domain is defined by the following equation:

f ₁(t)*f ₂(t)=∫_(−∞) ^(∞) f ₁(λ)f ₂(t−λ)dλ.

The value of the convolution for every time sample, t, is the sum overinfinity of the product of the two functions, but with the secondfunction offset by time t. The utility of the convolution operator liesin the fact that it is equivalent to multiplication in the frequencydomain:

Given the general notation:

F(e ^(jω))=the Fourier Transform of f(t)

if

f ₃(t)=f ₁(t)*f ₂(t)

then

F ₃(e ^(jω))=F ₁(e ^(jω))·F ₂(e ^(jω)).

A filter with a desired frequency response H(e^(jω)) can be implementedas a time domain operation, for example, by applying its inverse FourierTransform, h(t), in the convolution integral. In the present invention,however, the utility of the convolution operator is in the measurementof the time delay between two signals. In the simplest case, the twosignals are identical to one another, except that the second signal isdelayed by time t₀. As shown in the following equations, applying timedelay to a signal is equivalent to convolving that signal by the delayedimpulse function, δ(t−t₀):

f ₂(t)=f ₁(t−t ₀)

then

f ₂(t)=f ₁(t)*δ(t−t ₀).

Because convolution is associative, the problem of convolving the firstsignal f₁(t) with the second signal, f₂(t), can be solved by convolvingf₁(t) with itself and time delaying the result. Thus,

 f ₁(t)*f ₂(t)=f ₁(t)*f ₁(t)*δ(t−t ₀)

if

f ₃(t)=f ₁(t)*f ₁(t)

then

f ₁(t)*f ₂(t)=f ₃(t−t ₀).

Conversely, it is possible to measure the time delay between two signalsby convolving one with the other and detecting the amount of time delayin the result.

FIG. 39 illustrates the convolution of two similar signals 422 and 424,which are the inputs of a convolution operator 426 arriving at differenttimes. A correlogram 428 is a plot of the amplitude of the integratedproduct of signals 422 and 424. The horizontal axis of the correlogramrepresents the time delay applied to signal 422 relative to signal 424using the convolution operation, scaled in units of time samples. Adelay of around 400 samples is required to align signal 422 with signal424, which is evident from the peak value of the correlogram amplitude.Note that the correlogram is broader than either of the two signals.This condition can be understood from the recognition that convolving asignal with itself is equivalent to squaring the spectrum of the signal,a step that compresses the spectral distribution. Narrowing thebandwidth of a signal broadens the signal in the time domain.

In the velocity detection system, the signals 422 and 424 might havebeen generated by two objects traversing an optical grating 432, asshown in FIG. 40. A graph 430 represents the Gaussian illuminationprofile applied to the field of optical grating 432. An objecttraversing the illumination profile will generate a signal with aGaussian envelope and an oscillation frequency directly proportional tothe velocity of the object and inversely proportional to the opticalgrating pitch.

Detecting the peak of correlogram 428 might be accomplished using asimple peak detector. The correlogram is broad, however, and the peak ofthe envelope might not coincide with the peak of an oscillation cycle.More elaborate detection schemes might improve the accuracy, but it isalso useful to generate a narrower correlogram with a more clearlydefined peak. This objective is accomplished by using an optical grating436 shown in FIG. 41. Optical grating 436 has a non-uniform pitch, withline width (opaque and transparent bar width) decreasing linearly fromthe left end of the optical grating to the right end of the opticalgrating. The optical grating is aligned with a beam profile 434. Graphs438 and 440 in FIG. 42 represent signals that could be generated byphotodetectors in response to the light from two objects passing throughoptical grating 436. A resulting correlogram 442 is more compact thancorrelogram 428, suggesting that the time delay value might be extractedmore easily from correlogram 442 than from correlogram 428.

The preferred embodiment of the correlation-based signal generationsystem uses a detection system that takes advantage of the non-uniformoptical grating pitch. FIG. 43 illustrates the components used in thepreferred embodiment. Light source 12 and lens 36 illuminate the FOV offlow tube 16 for the purpose of velocity measurement. The optical systemcomprising lenses 40, 44, and 78 and beam splitter 76 form images of theobjects passing through the FOVs on two optical gratings 444 and 446having non-uniform but identical patterns of opaque and transparentbars.

As shown in FIG. 44, images 450 and 452 of optical gratings 444 and 446are aligned end-to-end along the axis of flow. The boundary between thetwo images is aligned with the midpoint of an illumination profile 454.Light scattered or emitted by objects in the flow stream is modulated byoptical gratings 444 and 446 and the modulated light is delivered tophotodetectors 50 and 50 a by lens 48 and a lens 448, respectively (seeFIG. 43).

FIG. 45 illustrates the performance of the correlation operation forsignals generated using the optical grating geometry shown in FIG. 44. Asignal 456, produced in response to light modulated by optical grating444 at the upstream side of the illumination field, grows in amplitudeand increases in frequency with time, and terminates when the objectmoves into the field of downstream side of the illumination field. Asignal 458, produced in response to light modulated by optical grating446 at the downstream side of the illumination field, starts at highamplitude and low frequency. The amplitude decays with time as thefrequency increases. A correlogram 460 shows a very distinct peak at theexact delay value that brings the two signals into alignment.

FIG. 46 shows an expanded view 462 of correlogram 460. For this view,the delay limits of the cross-correlation operation 426 were expanded toshow that as the delay of signal 458 approaches the delay for optimalalignment, it first passes through a region 466 in which the correlogramappears very noisy. This region is where the high-amplitude part ofsignal 456 is aligned with the high-amplitude part of signal 458.However, the particular optical grating configuration shown in FIG. 44provides the benefit that a primary peak 464 of correlogram 460 isbordered on both sides by very low-level signals 468. The noisy regionof the expanded correlogram is avoided by using only those delay valuesclose to the actual time of flight of the objects from a location on theupstream grating to the corresponding location on the downstreamgrating. A feedback loop in the supervisor program is used to regulatethe convolution time delay limits to maintain this condition.

FIG. 47 shows the functional processing blocks used for the signalacquisition and processing for this embodiment of the present invention.The signals respectively. The outputs of the amplifiers are applied tobandpass filters 54 and 54 a to eliminate DC bias and to preventaliasing when the signals are sampled by ADCs 56 and 56 a. The digitaloutputs of the ADCs are delivered to a signal processing stage 470,which accepts a signal segment of a predetermined length and delivers anestimate of the object velocity to a scrolling velocity list 472, ifacceptable signals from objects traversing the flow cell are present inthat segment. If a new velocity value is delivered by the signalprocessing operation, it is added to list 472 and the oldest value onthe list is deleted. A step 474 delivers the average of the velocityvalues in list 472 at the rate at which signal segments are captured inthe signal processor to facilitate computation of a running average 476.

FIG. 48 shows the detailed architecture of the signal processingoperation. For every cycle interval of the signal processor, concurrentsegments of the digitized signals from photodetectors 50 and 50 a arecaptured in steps 478 a and 478 b. The captured segments aresimultaneously applied to magnitude calculators 480 a and 480 b, and toa step 482, which provides for determining a cross-correlation. Eachmagnitude calculator uses the following algorithm for computing thesignal level:$M_{j} = {\sum\limits_{i = 1}^{N}\quad {{A\lbrack i\rbrack}}}$

where:

N=length of the signal segment

A[i]=value of the ith sample of the segment

M_(j)=magnitude of the jth signal segment.

The magnitude values are sent to supervisor program 486 to be used toregulate the photodetector amplifier gain.

The convolution (or cross-correlation) carried out in step 482 generatesthe correlogram using the following algorithm:

for (k=Min_Delay; k<=Max_Delay; k++)

{

m=k−Min_Delay;

for (i=0; i<=Correlation_Length; i++

{

j=k+i;

C[m]+=Signal 1[i]* Signal 2[j];

}

}

FIG. 49 illustrates the results of the correlation algorithm. A signalsegment 496 from the first photodetector is convolved with a signalsegment 498 from the second photodetector through a series ofmulti-and-accumulate operations to generate a correlogram 508. For eachvalue in correlogram 508, the first P samples, where P=CorrelationLength, of signal 496 are multiplied by the corresponding samples ofsignal 498 from sample Q, where Q=Delay, to sample R, whereR=Delay+Correlation Length. The products of the sample-by-samplemultiplication are summed to produce the values of the correlogram. TheDelay value begins at a Min_Delay 504 and advances one sample for everysample in correlogram 508 until it reaches a Max_Delay 506.

The location of the peak of the correlogram is found in a step 484 (seeFIG. 48) using a simple peak detection algorithm, as follows:

C_(pk)=0

for (d=1=>d=N)

if (C[d]>C_(pk))

then

[(C _(pk) =C[d]) and (N _(d) =d)]

where:

C[d]=value of correlogram at delay d

N_(d)=location of correlogram peak

C_(pk)=peak amplitude of correlogram.

In a step 488, the peak amplitude of the correlogram is compared to athreshold. This threshold is a fixed value accessible to a supervisorprogram 486. Regulation of the photodetector signal level using variablegain amplifiers 52 and 52 a enable the use of a fixed threshold.

If the peak amplitude of the correlogram exceeds the threshold, the peaklocation from step 484 is accepted and passed to a step 490 in which thevelocity is calculated. The velocity calculated in step 490 thenreplaces the oldest velocity value in scrolling velocity list 472 (seeFIG. 47). If the amplitude of the correlogram is less than or equal tothe threshold, the signal processor returns a NULL value 494, andscrolling velocity list 472 remains unchanged.

For a valid correlogram, a velocity 492 is computed from the correlogrampeak location using the following relation:

 t _(t) =N _(d) ·t _(samp)

and

v=s/t _(t)

where:

t_(t)=transit time, grating-to-grating (sec)

t_(samp)=signal sample time

s=grating-to-grating distance (mm)

v=velocity (mm/sec).

The running average velocity estimate is acquired by the supervisorprogram and translated into a frequency signal used by Instrument TimingGenerator 146, as shown in FIG. 19.

FIG. 50 shows the structure of the supervisor program for the fourthembodiment of the present invention. System operation is initiated in astart block 510 when the gain of the variable gain amplifier is set tonominal values, and objects are introduced into the flow stream forimage acquisition.

In a step 512, the supervisor program performs a cross-correlationbetween segments of the two photodetector signals using a wide span ofcorrelation delays. The delay value yielding the largest peak in thecorrelogram is used in a step 514 to compute the initial velocity. Thecross-correlation delay limits are set in a step 516 to bracket thisinitial delay value.

With the cross-correlation delay limits set, object velocity processingcommences. During this processing, the supervisor program continuouslymeasures the velocity using the cross correlation method and adjusts thecorrelation delay limits to maintain execution of a short spancross-correlation in the neighborhood of the delay required for thecurrent flow velocity. Use of the short span cross correlation reducescomputation time.

A step 478 a provides for capturing the photodetector signal segments; astep 482, computes the short delay span cross-correlation; a step 490computes the velocity and tabulates the results in a scrolling velocitylist 472 to provide the information for adjusting the correlationlimits. The limits are determined in a step 518 from the average of thevalues in velocity table 472. This average velocity is converted to anexpected correlation time delay value, and the limits are placedsymmetrically around this expected delay. The offset from the expectedvalue to the minimum delay and the offset from the expected value to themaximum delay are empirically determined and stored in a look-up tableto be used in the limit calculation step 518. In a step 516, thecorrelation offset limits are set and stored in locations accessible tothe cross-correlation determination in step 482 for use in processingthe next segment of the photodetector signals.

The supervisor program also continuously optimizes the gain of thevariable gain amplifiers to maximize SNR as specimen characteristicschange, without causing saturation at the ADC. The process of regulatingthe gains of the amplifiers is initiated in steps 480 a and 480 b, whichcompute the integrated magnitudes of the signal segments. The magnitudesare delivered to tables 520 and 526, which contain a set of the mostrecent magnitudes. Adjustments to the gains are computed by steps 522and 528 from the maximum magnitudes in tables 520 and 526 and the newsetting to variable gain amplifiers 52 and 52 a are made in steps 524and 530. The gain adjustment in steps 524 and 530 uses a “fast attack,”“slow recovery” algorithm as described above, to regulate the gain ofthe variable gain amplifiers.

It should be noted that the method described above for processing theelectrical signals produced by the photodetector(s) in the third andfourth embodiments using the TDVMs can also be applied to determiningthe velocity of objects disposed on a substrate (and the velocity of thesubstrate) that is caused to move through the FOV. Generally, either theTDVM or the FDVM approach can be used for determining the velocity ofany configuration of objects moving through the FOV.

Although the present invention has been described in connection with thepreferred form of practicing it, those of ordinary skill in the art willunderstand that many modifications can be made thereto within the scopeof the claims that follow. Accordingly, it is not intended that thescope of the invention in any way be limited by the above description,but instead be determined entirely by reference to the claims thatfollow.

The invention in which an exclusive right is claimed is defined by thefollowing:
 1. A velocity measurement system for determining anindication of a velocity of an object passing through a field of view,comprising: (a) an optical element disposed to direct light travelingfrom an object passing through the field of view along a collectionpath; (b) at least one optical grating of substantially uniform pitchdisposed in the collection path, said at least one optical gratingmodulating the light traveling along the collection path to producemodulated light having a modulation frequency proportional to a velocityof an object passing through the field of view; (c) at least one lightsensitive detector on which the modulated light is incident, said atleast one light sensitive detector producing an electrical signalresponsive to the modulated light; (d) means for processing theelectrical signal in the time domain to determine the indication of thevelocity.
 2. The measurement system of claim 1, wherein a pitch of theoptical grating is varied to compensate for distortion due to theoptical element.
 3. A velocity measurement system for determining anindication of a velocity of an object passing through a field of view,comprising: (a) an optical element disposed so that light traveling froman object passing through the field of view is directed along acollection path; (b) an optical grating disposed in the collection path,said optical grating modulating the light traveling along the collectionpath, to produce modulated light having a modulation frequencycorresponding to a velocity of an object passing through the field ofview; (c) a light sensitive detector disposed to receive the modulatedlight, said light sensitive detector responding to the modulated lightby producing a corresponding electrical signal; (d) means coupled tosaid light sensitive detector, for converting the electrical signal intoa sequence of digital samples; and (e) means in receipt of the sequenceof digital samples, for processing the sequence of digital samples inthe time domain to determine the indication of the velocity of anobject.
 4. The measurement system of claim 3, wherein said means forconverting the electrical signal into a sequence of digital samplescomprises an analog-to-digital converter.
 5. The measurement system ofclaim 3, further comprising an amplifier coupled to said light sensitivedetector, for amplifying the electrical signal before it is convertedinto the sequence of digital samples.
 6. The measurement system of claim3, further comprising a bandpass filter coupled to the light sensitivedetector, for filtering the electrical signal before it is convertedinto the sequence of digital samples.
 7. The measurement system of claim3, wherein said means for processing the sequence of digital samplescomprises at least one of a programmed computing device, an applicationspecific integrated circuit, and a digital oscilloscope.
 8. Themeasurement system of claim 3, further comprising a system controllerthat is coupled to and controls the means for converting the electricalsignal into the sequence of digital samples, and the means forprocessing the sequence of digital samples, said system controllercomprising at least one of a programmed computing device and anapplication specific integrated circuit.
 9. The measurement system ofclaim 8, wherein said system controller includes means for performing atleast one of the following functions: (a) regulating an amplitude ofsaid electrical signal; (b) determining a signal-to-noise ratio of theelectrical signal and rejecting any indication of the velocitydetermined if the signal-to-noise ratio is less than a predeterminedthreshold; and (c) regulating a local oscillation frequency employed bythe means for processing in response to variations in the indication ofthe velocity of an object that is determined.
 10. The measurement systemof claim 3 in which the light from an object passing through the fieldof view comprises at least one of light scattered by an object, anunstimulated emission from an object, and a stimulated emission from anobject.
 11. The measurement system of claim 3, wherein said opticalgrating comprises an optical grating having a substantially uniformpitch, and wherein said means for converting the electrical signal intoa sequence of digital samples comprises: (a) an amplifier having aninput that is coupled to said light sensitive detector and an output,said amplifier amplifying the electrical signal, produces an amplifiedelectrical signal at its output; (b) a bandpass filter coupled to theoutput of the amplifier, said bandpass filter filtering the amplifiedelectrical signal to produce a passband signal; and (c) a first analogbaseband converter and analog-to-digital converter that converts thepassband signal to a first sequence of digital samples, such that thefirst sequence of digital samples represents a real part of theelectrical signal; and (d) a second analog baseband converter andanalog-to-digital converter that converts the passband signal to asecond sequence of digital samples, such that the second sequence ofdigital samples represents an imaginary part of the electrical signal.12. The measurement system of claim 3, wherein said optical grating hasa uniform pitch, and wherein said means for processing the sequence ofdigital samples determines the indication of the velocity from a firstderivative of a time series of phase samples of the electrical signal,after calculating those phase samples from a baseband complexrepresentation of the electrical signal.
 13. The measurement system ofclaim 3, wherein said optical grating has a non uniform pitch.
 14. Themeasurement system of claim 13, wherein said optical grating comprises aplurality of alternating opaque bars and transparent gaps, widths ofwhich vary in a linear progression in a direction perpendicular to alongitudinal extent of the plurality of alternating opaque bars andtransparent gaps.
 15. The measurement system of claim 13, wherein saidoptical grating comprises a plurality of alternating opaque bars andtransparent gaps, widths of which vary in a non-linear pattern in adirection perpendicular to a longitudinal extent of the plurality ofalternating opaque bars and transparent gaps.
 16. The measurement systemof claim 13, wherein said optical grating comprises two substantiallyidentical sections, each section having a non-uniform pattern of bar andgap widths, disposed along an axis of motion of an object, such that anobject traverses said sections sequentially.
 17. The measurement systemof claim 13, further comprising: (a) a beam splitter that is disposed inthe collection path, such that a portion of the light traveling from anobject along the collection path is diverted along a second collectionpath; (b) another optical grating disposed in the second collectionpath, said other optical grating having a non uniform pitch andmodulating the light traveling along the second collection path, toproduce modulated light having a modulation frequency corresponding to avelocity of an object passing through the field of view; and (c) anotherlight sensitive detector disposed in the second collection path, saidother light sensitive detector converting the modulated light from saidat least one other optical grating into a second electrical signal. 18.The measurement system of claim 17, wherein said means for convertingthe electrical signal into a sequence of digital samples comprises: (a)a first amplifier having an input that is coupled to said at least onelight sensitive detector disposed in the collection path to receive theelectrical signal, and an output, said first amplifier amplifying theelectrical signal, producing an amplified electrical signal at itsoutput; (b) a first bandpass filter coupled to the output of the firstamplifier, said first bandpass filter filtering the amplified electricalsignal to produce a passband signal; (c) a first analog-to-digitalconverter that converts the passband signal to a first sequence ofdigital samples; (d) a second amplifier having an input that is coupledto said other light sensitive detector disposed in the second collectionpath, and an output, said second amplifier amplifying the secondelectrical signal, producing a second amplified electrical signal at itsoutput; (e) a second bandpass filter coupled to the output of the secondamplifier, said second bandpass filter filtering the second amplifiedelectrical signal to produce a second passband signal; and (f) a secondanalog-to-digital converter that converts the second passband signal toa second sequence of digital samples.
 19. The measurement system ofclaim 17, wherein said means for converting the electrical signal into asequence of digital samples comprises: (a) a first amplifier having aninput that is coupled to said light sensitive detector disposed in thecollection path, and an output, said first amplifier amplifying theelectrical signal, producing an amplified electrical signal at itsoutput; (b) a first analog-to-digital converter that converts theamplified electrical signal to a first sequence of digital samples; (c)a first digital bandpass filter for rejecting a direct current bias anda high frequency noise component from the first sequence of digitalsamples; (d) a second amplifier having an input that is coupled to saidother light sensitive detector disposed in the second collection path,and an output, said second amplifier amplifying the second electricalsignal, producing a second amplified electrical signal at its output;(e) a second analog-to-digital converter that converts the secondamplified electrical signal to a second sequence of digital samples; and(f) a second digital bandpass filter for rejecting a direct current biasand a high frequency noise component from the second sequence of digitalsamples.
 20. The measurement system of claim 17, wherein each opticalgrating is aligned in series along an axis of motion of an object, andwherein said means for processing the sequence of digital samples todetermine the indication of the velocity of an object calculates anamplitude peak of a cross-correlogram generated by a convolution of theelectrical signal from said light sensitive detector disposed in thecollection path with the second electrical signal from said other lightsensitive detector disposed in the second collection path.
 21. Themeasurement system of claim 17, further comprising a control systemcontrollably connected to said means for converting the electricalsignal into a sequence of digital samples and said means for processingthe sequence of digital samples.
 22. The measurement system of claim 21,wherein said control system regulates: (a) a gain of the first amplifierand the second amplifier in response to varying levels of the electricalsignal and the second electrical signal, respectively; and (b) an upperand a lower limit of time shifting in a cross-correlation operation inresponse to variations in the velocity of an object.
 23. The measurementsystem of claim 3, further comprising at least one light source forilluminating the field of view.
 24. The measurement system of claim 23,wherein said at least one light source is disposed to provide anincident light that illuminates an object passing through the field ofview.
 25. The measurement system of claim 24, wherein said incidentlight stimulates at least one of an emission and a fluorescence from anobject passing through the field of view, and the light from the objectpassing through the field of view comprises one of an emitted light anda fluoresced light.
 26. The measurement system of claim 24, wherein theincident light is at least partially absorbed by an object passingthrough the field of view, so that the light from an object has beenchanged due to the absorption by an object.
 27. The measurement systemof claim 24, wherein the light is reflected from an object passingthrough the field of view toward the optical element.
 28. Themeasurement system of claim 23, wherein the light source comprises atleast one of a coherent light source, a non-coherent light source, apulsed light source, a continuous light source, a continuous wave laser,a pulsed laser, a continuous wave incandescent lamp, a strobe arc lamp,and an optical filter that provides a limited spectrum of light forilluminating an object.
 29. The measurement system of claim 3, furthercomprising a source of a reference field light, wherein an intensity ofthe light from an object passing through the field of view is modulatedby a phase interference with the reference field light.
 30. Themeasurement system of claim 3, wherein a flow of a fluid in whichobjects are entrained passes through the field of view, such that anindication of a velocity of an object entrained in the fluid isdetermined by the means for processing.
 31. The measurement system ofclaim 3, wherein a support on which a plurality of objects are disposedpasses through the field of view, such that an indication of a velocityof an object on the support and thus, of the support, is determined bythe means for processing.
 32. The measurement system of claim 3, whereinsaid optical element comprises a lens.
 33. The measurement system ofclaim 3, wherein the other light sensitive detector and a dispersingelement that directs the light from the optical element to each of thelight sensitive detectors.
 34. The measurement system of claim 33,wherein the other light sensitive detector is employed to determine acharacteristic of an object passing through the field of view, otherthan an indication of a velocity of an object.
 35. The measurementsystem of claim 3, wherein an object passes through another field ofview, further comprising: (a) another optical element disposed to directlight from an object passing through the other field of view alonganother collection path; and (b) another light sensitive detectordisposed to receive the light traveling along the other collection pathand employed to determine a characteristic of an object passing throughthe other field of view, said characteristic being other than theindication of the velocity of an object.
 36. The measurement system ofclaim 3, further comprising means disposed downstream from said field ofview, for sorting objects.
 37. The measurement system of claim 3,wherein said light sensitive detector comprises one of a photosensitivediode and a photomultiplier tube.
 38. The measurement system of claim 3,further comprising a fluid supply disposed upstream of the field ofview, said fluid supply providing the field of view with a flow of afluid in which a plurality of objects are entrained.
 39. The measurementsystem of claim 38, wherein each of the plurality of objects comprisesone of a biological cell and a particulate component of a biologicalspecimen.
 40. The measurement system of claim 3, further comprising asolid support on which a plurality of objects are disposed, and a primemover, said solid support being moved through the field of view by theprime mover.
 41. An optical analysis system employed to determine anindication of a velocity of a relative movement between an object andthe optical analysis system, and to determine at least one additionalcharacteristic of the object, comprising: (a) a first optical elementdisposed to direct light from an object along a first collection path;(b) a second optical element disposed in the first collection path todirect a portion of the light traveling along the first collection path,along a second collection path; (c) an optical grating disposed in thesecond collection path, said optical grating modulating the lighttraveling along the second collection path, producing modulated lightthat has a modulation frequency corresponding to a velocity of therelative movement between the object and the optical analysis system;(d) a light sensitive detector disposed in the second collection path toreceive the modulated light, said at least one light sensitive detectorproducing an electrical signal in response to the modulated light; (e)means coupled to the light sensitive detector to receive the electricalsignal, for determining the indication of the velocity of the relativemovement between the object and the optical analysis system as afunction of the electrical signal and producing a timing signal as afunction of said indication of the velocity; (f) a time delayintegration (TDI) detector disposed to receive light traveling along thefirst collection path, said TDI detector being coupled to said means fordetermining the indication of the velocity, said TDI detector employingthe timing signal to produce an output signal that is indicative of saidat least one additional characteristic of the object.
 42. The opticalanalysis system of claim 41, further comprising a control that iscoupled to and controls the means for determining the indication of thevelocity, and the TDI detector.
 43. The optical analysis system of claim41, wherein said optical grating has a substantially uniform pitch. 44.The optical analysis system of claim 43, wherein said means fordetermining the indication of the velocity of the relative movementbetween the object and the optical analysis system comprises: (a) anamplifier having an input that is coupled to the light sensitivedetector disposed in the second collector path and an output, saidamplifier amplifying the electrical signal, producing an amplifiedelectrical signal at its output; (b) an analog-to-digital converter thatconverts the amplified electrical signal to a sequence of digitalsamples; and (c) processing means for processing the sequence of digitalsamples to determine the indication of the velocity of the object. 45.The optical analysis system of claim 43, wherein said means fordetermining the indication of the velocity of the relative movementbetween the object and the optical analysis system comprises: (a) anamplifier having an input that is coupled to the light sensitivedetector disposed in the second collector path and an output, saidamplifier amplifying the electrical signal, producing an amplifiedelectrical signal at its output; (b) a bandpass filter coupled to theoutput of the amplifier, said bandpass filter filtering the amplifiedelectrical signal to produce a passband signal; and (c) a first analogbaseband converter and analog-to-digital converter that converts thepassband signal to a first sequence of digital samples, such that thefirst sequence of digital samples represents a real part of theelectrical signal, and (d) a second analog baseband converter andanalog-to-digital converter that converts a portion of the passbandsignal into a second sequence of digital samples, such that the secondsequence of digital samples represents an imaginary part of theelectrical signal, and (e) processing means for processing the first andsecond sequences of digital samples to determine the indication of thevelocity of the relative movement between the object and the opticalanalysis system.
 46. The optical analysis system of claim 44, whereinsaid processing means calculates a time series of phase samples from abaseband complex representation that is derived from the sequence ofdigital samples.
 47. The optical analysis system of claim 41, whereinsaid optical grating disposed in the second collection path has asubstantially non uniform pitch.
 48. The optical system of claim 47,wherein said optical grating disposed in the second collection pathcomprises at least one of: (a) a plurality of alternating opaque barsand transparent gaps, widths of which vary in a linear progression in adirection perpendicular to a longitudinal extent of the plurality ofalternating opaque bars and transparent gaps; and (b) a plurality ofalternating opaque bars and transparent gaps, widths of which vary in anon-linear pattern in a direction perpendicular to a longitudinal extentof the plurality of alternating opaque bars and transparent gaps. 49.The optical analysis system of claim 47, wherein the optical gratingdisposed in the second collection path and having the substantially nonuniform pitch comprises two substantially identical sections, eachhaving a non-uniform pattern of bar and gap widths and disposed along anaxis of motion of light from the object such that the light from theobject traverses said sections sequentially.
 50. The optical analysissystem of claim 47, further comprising: (a) a third optical elementdisposed in the second collection path, such that a portion of the lighttraveling from the object along the second collection path is divertedalong a third collection path; (b) an optical grating disposed in thethird collection path and having a substantially non uniform pitch, saidoptical grating modulating the light, to produce modulated light havinga modulation frequency corresponding to the velocity of the objectpassing through the field of view; and (c) a light sensitive detectordisposed in the third collection path and used to convert the modulatedlight traveling along the third collection path into a second electricalsignal, said light sensitive detector being coupled to provide thesecond electrical signal to said means for determining the indication ofthe velocity of the relative movement between the object and the opticalanalysis system.
 51. The optical analysis system of claim 50, whereinsaid means for determining the indication of the velocity of therelative movement between the object and the optical analysis systemcomprises: (a) a first amplifier having an input that is coupled to thelight sensitive detector disposed in the second collection path, and anoutput, said first amplifier amplifying the electrical signal, producingan amplified electrical signal at its output; (b) a first bandpassfilter coupled to the output of the first amplifier, said first bandpassfilter filtering the amplified electrical signal provided by the firstamplifier to produce a passband signal; (c) a first analog-to-digitalconverter that converts the passband signal to a first sequence ofdigital samples; (d) a second amplifier having an input that is coupledto the light sensitive detector disposed in the third collection path,and an output, said second amplifier amplifying the second electricalsignal, producing a second amplified electrical signal at its output;(e) a second bandpass filter coupled to the output of the secondamplifier, said second bandpass filter filtering the second amplifiedelectrical signal to produce a second passband signal; (f) a secondanalog-to-digital converter that converts the second passband signal toa second sequence of digital samples; and (g) processing means forprocessing each sequence of digital samples, to determine the indicationof the velocity of the relative movement between the object and theoptical analysis system.
 52. The optical analysis system of claim 50,wherein said means for determining the indication of the velocity of therelative movement between the object and the optical analysis systemcomprises: (a) a first amplifier having an input that is coupled to thelight sensitive detector disposed in the second collection path, and anoutput, said first amplifier amplifying the electrical signal, producinga first amplified electrical signal at its output; (b) a firstanalog-to-digital converter that converts the first amplified electricalsignal to a first sequence of digital samples; (c) a first digitalbandpass filter for rejecting a direct current bias and a high frequencynoise component from the first sequence of digital samples; (d) a secondamplifier having an input that is coupled to the light sensitivedetector disposed in the third collection path, and an output, saidsecond amplifier amplifying the second electrical signal, producing asecond amplified electrical signal at its output; (e) a secondanalog-to-digital converter that converts the second amplifiedelectrical signal to a second sequence of digital samples; (f) a seconddigital bandpass filter for rejecting a direct current bias and a highfrequency noise component from the second sequence of digital samples;(g) processing means for processing each sequence of digital samples, todetermine the indication of the velocity between the object and theoptical analysis system.
 53. The optical analysis system of claim 50,wherein said means for determining the indication of the velocity of therelative movement between the object and the optical analysis systemdetermines an amplitude peak of a cross-correlogram generated by aconvolution of the electrical signal from the light sensitive detectordisposed in the second collection path with the second electrical signalfrom the light sensitive detector disposed in the third collection path,so that an image field associated with the optical grating disposed inthe second collection path and a second image field associated with theoptical grating disposed in the third collection path are aligned inseries along an axis of the relative movement between the object and theoptical analysis system.
 54. The optical analysis system of claim 51,wherein said processing means comprises at least one of a digitaloscilloscope, a programmed computing device, and an application specificintegrated circuit.
 55. The optical analysis system of claim 52, furthercomprising a control system controllably connected to each amplifier andto said processing means, said control system regulating an upper and alower limit of a time shifting employed in determining across-correlation in response to variations in the velocity of therelative movement between the object and the optical analysis system.56. An imaging system adapted to determine an indication of a velocityof a relative movement between the imaging system and an object, and atleast one additional characteristic of the object, comprising: (a) anoptical element disposed to direct light traveling from an object alonga first collection path; (b) a beam splitter disposed in the firstcollection path so that a portion of the light traveling from the objectalong the first collection path is diverted along a second collectionpath; (c) an optical grating disposed in the second collection path, theoptical grating modulating the collected light traveling along thesecond collection path, to produce modulated light having a modulationfrequency corresponding to the velocity of the relative movement betweenthe object and the imaging system; (d) a light sensitive detectordisposed in the second collection path to receive the modulated light,said light sensitive detector producing an electrical signal in responseto the modulated light received along the second collection path; (e)means coupled to the light sensitive detector, for determining theindication of the velocity of the relative movement between the objectand the imaging system as a function of the electrical signal, saidmeans producing a timing signal as a function of said indication of thevelocity; (f) an imaging lens disposed in the first collection path,producing an image from the light traveling along the first collectionpath; and (g) a time delay integration (IDI) detector disposed in thefirst collection path to receive the image produced by the imaging lens,producing an output signal that is indicative of said at least oneadditional characteristic of the object, said TDI detector being coupledto the means for determining the indication of the velocity to receivethe timing signal and producing the output signal by using the timingsignal to provide a clocking function that synchronizes the TDI detectorto a movement of the image of the object over the TDI detector so that aresponse to the light comprising the image is integrated over time asthe image moves over the TDI detector.
 57. An imaging system fordetermining an indication of a relative velocity and at least oneadditional characteristic of an object, from images of the object, whilethere is relative movement between the object and the imaging system,comprising: (a) a light source that emits light illuminating the object;(b) an optical element disposed to direct light from the object along afirst collection path; (c) a first beam splitter disposed in the firstcollection path so that light scattered from the object is directedalong a different path, while light that is emitted by the objectcontinues through the beam splitter along the first collection path; (d)a second beam splitter disposed in the first collection path so that aportion of the light from the object traveling along the firstcollection path is diverted along a second collection path; (e) anoptical grating disposed in the second collection path, said opticalgrating modulating the light traveling along the second collection path,to produce modulated light having a modulation frequency correspondingto the velocity of the relative movement between the object and theimaging system; (f) a light sensitive detector disposed to receive themodulated light, producing an electrical signal in response thereto; (g)means coupled to the light sensitive detector to receive the electricalsignal, for determining the indication of the relative velocity betweenthe object and the imaging system and producing a timing signal as afunction of said indication of the relative velocity; (h) a spectraldispersing element disposed downstream of the second beam splitter inthe first collection path, said spectral dispersing element spectrallydispersing the light emitted by the object, producing spectrallydispersed light; (i) an optical element disposed to receive thespectrally dispersed light, producing an image thereof; (j) acylindrical lens disposed to receive the light scattered from theobject, said cylindrical lens having a central axis around which thecylindrical lens is curved, said central axis being generally orthogonalto a direction of the relative movement between the object and theimaging system, so that the cylindrical lens produces a scatteredpattern image of the object along a direction that is substantiallyparallel to said central axis of the cylindrical lens; and (k) at leastone time delay integration (TDI) detector disposed to receive the image,producing an output signal that is indicative of at least onecharacteristic of the object, and to receive the scattered pattern imageproduced by the cylindrical lens, producing a different output signalthat is indicative of at least one other characteristic of the object,said at least one TDI detector being coupled to the means fordetermining the indication of the relative velocity of the movementbetween the object and the imaging system, said at least one TDIdetector producing said output signals by using the timing signal forsynchronization of said output signals with movement of the image andthe scattered pattern image over said at least one TDI detector.
 58. Anoptical analysis system employed to determine an indication of avelocity of a relative movement between an object and the opticalanalysis system and to determine and at least one other characteristicof the object, comprising: (a) a first collection optical elementdisposed so that light from the object is directed along a firstcollection path; (b) a first light dispersing optical element disposedin the first collection path so as to disperse the light traveling alongthe first collection path, producing first dispersed light; (c) a firstimaging optical element disposed to receive the first dispersed light,forming at least one image from the first dispersed light; (d) a secondcollection optical element disposed to direct light from the objectalong a second collection path, different than the first collectionpath; (e) a second light dispersing optical element disposed in thesecond collection path, so as to disperse the light traveling along thesecond collection path, producing second dispersed light; (f) a secondimaging optical element disposed to receive the second dispersed light,forming at least one image from said second dispersed light; (g) a beamsplitter that is disposed in one of the first collection path and thesecond collection path, so that a portion of the light traveling fromthe object along said one of the first collection path and the secondcollection path is diverted along a third collection path; (h) anoptical grating disposed in the third collection path, the opticalgrating modulating the light traveling along the third collection path,to produce modulated light having a modulation frequency correspondingto the velocity of the relative movement between the object and theoptical analysis system; (i) a light sensitive detector disposed in thethird collection path to receive the modulated light, the lightsensitive detector producing an electrical signal in response to themodulated light; (j) means coupled to the light sensitive detector toreceive the electrical signal, for determining the indication of thevelocity of the relative movement between the object and the opticalanalysis system, producing a timing signal corresponding thereto; (k) afirst time delay integration (TDI) detector coupled to the means fordetermining the indication of the velocity to receive the timing signaland disposed to receive said at least one image formed by the firstimaging optical element, said first TDI detector producing a firstoutput signal that is indicative of at least one characteristic of theobject by integrating said at least one image formed from the firstdispersed light over time using the timing signal for synchronization ofthe first TDI detector with the movement over the first TDI detector ofsaid at least one image formed by the first imaging optical element; and(l) a second TDI detector disposed to receive said at least one imageformed by the second imaging optical element, said second TDI detectorbeing coupled to said means for determining the indication of thevelocity to receive the timing signal and producing a second outputsignal that is indicative of at least one other characteristic of themoving object by integrating said at least one image formed from thesecond dispersed light over time using the timing signal forsynchronization of the second TDI detector with the relative motion ofsaid at least one image formed by the second imaging optical element.59. A method for determining an indication of a velocity of an object inmotion using light from the object, comprising the steps of: (a)modulating light from the object using an optical grating to producemodulated light having a modulation frequency that varies as a functionof the velocity of the object; (b) producing an electrical signalcorresponding to an intensity of the modulated light; (c) processing theelectrical signal in the time domain to determine the indication of thevelocity of the object.
 60. The method of claim 59, further comprisingthe step of amplifying the electrical signal before the step ofprocessing.
 61. The method of claim 59, further comprising the step offiltering the electrical signal before the step of processing.
 62. Themethod of claim 61, wherein the step of filtering the electrical signalcomprises the step of: (a) removing a direct current bias from theelectrical signal; and (b) eliminating frequencies above a predeterminedNyquist limit from the electrical signal.
 63. The method of claim 59,wherein the step of modulating light from the object using an opticalgrating comprises the step of utilizing an optical grating having asubstantially uniform pattern.
 64. The method of claim 63, furthercomprising the steps of: (a) amplifying the electrical signal before thestep of processing, producing an amplified signal; (b) converting theamplified signal into a complex analog baseband signal; (c) convertingthe complex analog baseband signal into a first digital sample sequencerepresenting a real part of the amplified signal; and (d) converting thecomplex analog baseband signal into a second digital sample sequencerepresenting an imaginary part of the amplified signal.
 65. The methodof claim 63, further comprising the steps of: (a) amplifying theelectrical signal before the step of processing, producing an amplifiedsignal; (b) filtering the amplified signal, producing a passband signal;(c) converting the passband signal into a complex analog basebandsignal; (d) converting the complex analog baseband signal into a firstdigital sample sequence representing a real part of the passband signal;and (e) converting the complex analog baseband signal into a seconddigital sample sequence representing an imaginary part of the passbandsignal.
 66. The method of claim 63, wherein the step of processingcomprises the steps of: (a) producing a baseband complex representationof the electrical signal; (b) phase sampling the baseband complexrepresentation of the electrical signal to determine a time series ofphase samples of the baseband complex representation; and (c)determining a first derivative of the time series of the phase samplesto determine the indication of the velocity of the object.
 67. Themethod of claim 63, wherein the step of processing comprises the stepsof: (a) sampling the electrical signal, producing a sequence of digitalsamples; (b) splitting the sequence of digital samples into a pluralityof baseband signal pairs; (c) separating the plurality of basebandsignal pairs into sample packets, according to predefined criteria; (d)applying an unwrapping algorithm to each sample packet; (e) rejectingsample packets having a width outside of predefined limits; and (f)determining the indication of the velocity of the object based upon asample packet that is not rejected.
 68. The method of claim 67, furthercomprising the step of applying a lowpass filter to each baseband signalpair before the step of separating the baseband signal pairs into samplepackets.
 69. The method of claim 67, further comprising the step ofemploying a sequence of upper and lower sideband samples derived fromthe sequence of complex baseband samples for automated control of themethod.
 70. The method of claim 63, further comprising the steps of: (a)applying a Hilbert transform to the electrical signal before the digitalsamples; and (b) using the upper sideband and the lower sideband digitalsamples to: (i) determine a frequency associated with a light sensitivedetector; and (ii) setting a local oscillator frequency for a basebanddemodulator used for determining the indication of the velocity of theobject.
 71. The method of claim 59, wherein the step of processingcomprises the step of utilizing at least one of a digital oscilloscope,a programmed computing device, and an application specific integratedcircuit to perform the processing.
 72. The method of claim 59, furthercomprising the steps of: (a) providing a control system; and (b) usingthe control system to perform at least one of the following functions:(i) regulating an amplification gain applied to the electrical signal inresponse to a varying level of the electrical signal; (ii) determining asignal-to-noise ratio of the electrical signal for use in regulating athreshold applied to frequency measurements; and (iii) regulating afrequency of a baseband converter local oscillator used in the step ofprocessing, as a function of the velocity of the object.
 73. The methodof claim 59, wherein the step of modulating light from the object usingan optical grating comprises the step of utilizing an optical gratinghaving a substantially non uniform pattern.
 74. The method of claim 73,wherein the step of utilizing an optical grating comprises the step ofutilizing an optical grating having at least one of: (a) a plurality ofalternating opaque bars and transparent gaps, widths of which vary in alinear progression in a direction perpendicular to a longitudinal extentof the bars and the transparent gaps; and (b) a plurality of alternatingopaque bars and transparent gaps, the width of which vary in anon-linear pattern in a direction perpendicular to a longitudinal extentof the bars and the transparent gaps.
 75. The method of claim 73,wherein the step of utilizing an optical grating comprises the step ofutilizing an optical grating having two substantially identicalsections, each with a substantially non-uniform pattern of bar and gapwidths, disposed along an axis of motion of the object such that lightfrom the object sequentially traverses said sections.
 76. The method ofclaim 59, wherein the step of modulating light from the object using anoptical grating comprises the steps of utilizing a first optical gratingto modulate a first light beam from the object, producing firstmodulated light; and utilizing a second optical grating to modulate asecond light beam from the object, producing second modulated light,wherein the first and second optical gratings have a substantially nonuniform pattern.
 77. The method of claim 76, wherein the step ofmodulating light from the object using an optical grating furthercomprises the step of aligning the first and second optical gratingssuch that image fields of the first and second optical gratings aredisposed in series along an axis of motion of the object.
 78. The methodof claim 76, wherein the step of producing an electrical signalcorresponding to an intensity of the modulated light comprises the stepsof producing a first electrical signal corresponding to the firstmodulated light, and producing a second electrical signal correspondingto the second modulated light, further comprising the steps ofconverting the first electrical signal into a first sequence of digitalsamples, and converting the second electrical signal into a secondsequence of digital samples.
 79. The method of claim 78, furthercomprising the step of amplifying the first electrical signal and thesecond electrical signal before converting the first electrical signaland the second electrical signal into the first and second sequences ofdigital samples.
 80. The method of claim 78, further comprising the stepof filtering the first electrical signal and the second electricalsignal before converting the first electrical signal and secondelectrical signal into the first sequence and second sequence of digitalsamples, respectively.
 81. The method of claim 76, wherein the step ofprocessing comprises the steps of: (a) convolving the first electricalsignal with the second electrical signal to produce a convolution; (b)generating a cross-correlogram based on the convolution; and (c)determining the indication of the velocity from a peak amplitude of thecross-correlogram.
 82. The method of claim 76, further comprising thesteps of: (a) providing a control system; and (b) using the controlsystem to regulate an upper and a lower limit of a time shift in across-correlation function in response to a variation in the velocity ofthe object.
 83. A method for determining an indication of a relativevelocity associated with an object and one or more other characteristicsof an object, comprising the steps of: (a) directing light from theobject along a first collection path; (b) diverting a portion of thelight traveling along the first collection path to a second collectionpath that is in a different direction than the first collection path;(c) modulating light traveling along the second collection path as afunction of the relative velocity; (d) producing an electrical signal asa function of an intensity of the modulated light; (e) processing theelectrical signal in the time domain to determine the indication of therelative velocity associated with the object; (f) providing a time delayintegration (TDI) detector disposed to receive the light that istraveling along the first collection path; (g) using the indication ofthe relative velocity associated with the object to synchronize the TDIdetector with a motion of an image of the object on the TDI detector;and (h) analyzing an output signal from the TDI detector to determine atleast one other characteristic of the object.
 84. The method of claim83, wherein the step of modulating the light comprises the step ofproviding an optical grating having a substantially uniform pitch; andwherein the step of processing the electrical signal to determine theindication of the relative velocity comprises the steps of: (a)producing a baseband complex representation of the electrical signal;(b) producing a time series of phase samples from the baseband complexrepresentation; and (c) determining the indication of the relativevelocity from a first derivative of the time series of phase samples.85. The method of claim 83, wherein the step of modulating the lightcomprises the step of providing an optical grating that modulates thelight, further comprising the steps of: (a) diverting a portion of thelight traveling along the second collection path to a third collectionpath that is in a different direction than the first and secondcollection paths, (b) providing a second optical grating having asubstantially non uniform pitch, the second optical grating beingdisposed along the third collection path such that the optical gratingand the second optical grating are aligned in series along an axis ofrelative motion associated with the object, the second optical gratingmodulating light traveling along the third collection path; and (c)producing a second electrical signal as a function of an intensity ofthe modulated light traveling along the third collection path, whereinthe step of processing the electrical signal to determine the indicationof the relative velocity associated with the object comprises the stepsof: (i) convolving the electrical signal with the second electricalsignal to form a convolution; (ii) producing a cross-correlogram basedon the convolution; and (iii) determining the indication of the relativevelocity from a peak amplitude of the cross-correlogram.
 86. A methodfor determining an indication of a velocity and one or more othercharacteristics associated with a moving object, while there is relativemovement between the object and an optical system used to determine theindication of the velocity and the one or more other characteristics,comprising the steps of: (a) directing light from the object along afirst collection path and a second collection path, the first and secondcollection paths being in a different direction than a direction of therelative movement between the object and the optical system; (b)modulating light traveling along the first collection path; (c)producing an electrical signal as a function of an intensity of themodulated light; (d) processing the electrical signal in the time domainto determine the indication of the relative velocity between the opticalsystem and the object; (e) dispersing the light that is traveling alongthe second collection path, producing dispersed light; (f) providing atime delay integration (TDI) detector disposed to receive the dispersedlight; (g) using the indication of the relative velocity to synchronizethe TDI detector with movement of the dispersed light from the objectover the TDI detector; and (h) analyzing an output signal from the TDIdetector to determine said one or more other characteristics of theobject.
 87. The method of claim 86, wherein the step of modulatingcomprises the step of providing an optical grating having asubstantially uniform pitch, and wherein the step of processing theelectrical signal to determine the indication of the velocity betweenthe optical system and the object comprises the steps of: (a) creating abaseband complex representation of the electrical signal; (b) producinga time series of phase samples from the baseband complex representationof the electrical signal; and (c) determining a first derivative of thetime series of phase samples to determine the indication of the velocitybetween the optical system and the object.
 88. The method of claim 86,further comprising the steps of: (a) diverting a portion of the lighttraveling along the first collection path to a third collection paththat is in a different direction than the first and second collectionpaths, (b) modulating light traveling along the third collection path,producing modulated light; (c) producing a second electrical signal as afunction of an intensity of the modulated light traveling along thethird collection path; (d) convolving the electrical signal and thesecond electrical signal to produce a cross-correlogram; and (e)determining the indication of the velocity between the optical systemand the object from a peak amplitude of the cross-correlogram.